Microgel particles for the delivery of bioactive materials

ABSTRACT

Novel microgels, microparticles and related polymeric materials capable of delivering bioactive materials to cells for use as vaccines or therapeutic agents. The materials are made using a crosslinker molecule that contains a linkage cleavable under mild acidic conditions. The crosslinker molecule is exemplified by a bisacryloyl acetal crosslinker. The new materials have the common characteristic of being able to degrade by acid hydrolysis under conditions commonly found within the endosomal or lysosomal compartments of cells thereby releasing their payload within the cell. The materials can also be used for the delivery of therapeutics to the acidic regions of tumors and sites of inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/368,576, which was filed on Mar. 29, 2002, which is incorporatedby reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made during work partially supported by the U.S.Department of Energy under Contract No. DE-AC03-76SF00098. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to the field of cross-linked hydrogelpolymers formed into microgels for use in delivery of bioactivematerials such as antigens, DNA and other therapeutics.

2. Description of the Related Art

There is a great need for the development of vaccines against AIDS,Hepatitis C, cancer and other diseases. Traditional vaccinationstrategies, based on live attenuated viruses, have been ineffective atgenerating vaccines against these diseases, largely as result of theirhigh toxicity. Vaccines based on protein antigens are a new vaccinationstrategy that have considerable promise because of their low toxicityand widespread applicability. However, the clinical success ofprotein-based vaccines has been limited, due to delivery problems, andnew protein delivery vehicles are needed that can enhance the efficacyof protein-based vaccines.

A key limitation of current protein-based vaccines is their inability toactivate cytotoxic T lymphocytes (CTL). The activation of CTL iscritical for the development of immunity against viruses and tumors. CTLare activated by antigen presenting cells (APCs) through the Class Iantigen presentation pathway. APCs generally only present cytoplasmicproteins as Class I antigens, although Class I antigen presentation ofproteins residing in phagosomes also occurs under certain circumstances(Jondal, M., Schirmbeck, R. & Reimann, J. (1996) Immunity 5, 295–302.

Microparticles, 0.2–5 μm in diameter, have recently gained interest asdelivery vehicles for protein-based vaccines because of their ability toenhance the Class I antigen presentation of protein antigens (Oh,Yu-Kyoung, Harding, C. V.; Swanson, J. A.; Vaccine. 1997, (15), 511–518;Andrianov, et al., U.S. Pat. No. 5,529,777; and Staas, et al., U.S. Pat.No. 6,321,731). Two mechanisms have been proposed to explain the abilityof microparticles to enhance the Class I antigen presentation of proteinantigens. The first involves disruption of phagosomes by microparticlesleading to release of protein antigens into the cytoplasm of APCs, wherethey are processed for antigen presentation as endogenous proteins. Thesecond uses microparticles to deliver protein antigens to phagolysosomalcompartments that contain MHC I receptors That are being recycled fromthe plasma membrane. Once delivered these proteins are subsequentlydegraded by phagolysosomal enzymes into antigenic peptides that complexMHC I receptors and are then trafficked to the cell surface for antigenpresentation.

Protein therapeutics have tremendous clinical potential and arecurrently being investigated for the treatment of cancer, vaccinedevelopment and for manipulating the host response to implantedbiomaterials. However, the effective utilization of protein therapeuticsrequires the development of materials that can deliver bioactivematerial to diseased tissues and cells. At present, the majority ofprotein delivery vehicles are based on hydrophobic polymers, such aspoly(lactide-co-glycolide) (PLGA). See O'Hagan, D. et al., in U.S. Pat.Nos. 6,306,405 and 6,086,901, and in Adv. Drug Delivery Rev, 32, 225(1998). However, PLGA based delivery vehicles have been problematicbecause of their poor water solubility. Proteins are encapsulated intoPLGA based materials through an emulsion procedure that exposes them toorganic solvents, high shear stress and/or ultrasonic cavitation. Thisprocedure frequently causes protein denaturation and inactivation asshown by Xing D et al., Vaccine, 14, 205–213 (1996). Hydrogels andmicrogels have therefore been proposed as an alternative proteindelivery vehicle because they can encapsulate the protein in a totallyaqueous environment, under mild conditions. See Park, K. et al.,Biodegradable Hydrogels for Drug Delivery; Technomic Publishing Co,Lancaster, Pa. (1993); Peppas. N. A. Hydrogels in Medicine and Pharmacy;CRC press: Vol II, Boca Raton, Fla., 1986; and Lee, K. Y. et al.,Chemical Reviews, 101, 1869-179 (2001).

A key problem in the field of hydrogel research is the development ofmaterials that can release their contents in response to pathologicalstimuli, allowing for the targeting of protein therapeutics to diseasedtissues and cells. A particularly important pathological stimulus ismildly acidic pH. For example, tumors exist at acidic pHs between6.4–6.8, and the phagolysosomes of phagocytic cells are at pHs between4.5–5.0. The acidic nature of these compartments has stimulated a needfor the development of hydrogels and microgels that can selectivelyrelease their contents under mildly acidic conditions.

A particularly important application of protein delivery systems is thedevelopment of particulate materials that can deliver proteins tophagocytic cells, such as antigen presenting cells. Micron sized proteinloaded hydrogels (microgels) have been investigated for this purposebecause they are small enough to be phagocytosed. At present, micronsized hydrogels have been synthesized using crosslinkers that do notdegrade under biological conditions, and hence have had limited successin drug delivery applications.

Currently, hydrogels are synthesized using crosslinkers that containeither, amide, ester, or carbonate linkages. Sawhney, A. et al.,Macromolecules, 26, 581–587 (1993), describe bioerodible hydrogels basedon photopolymerized poly(ethyleneglycol)-co-poly(α-hydroxy acid)diacrylate macromers which utilize an ester linkage. Sheppard, R. C. etal., in U.S. Pat. No. 5,191,015, describe an insoluble polymer withcontiguous cleavable crosslinkers and functional groups, wherein thecrosslinking agent is an acid degradable ketal crosslinker. Sanxia. L,et al., describe release behavior of high molecular weight solutes frompoly(ethylene glycol)-based degradable networks in Macromolecules, 33,2509–2515 (2000). See also Dijk-Wolthius, W. N. E. et al.,Macromolecules. 30, 4639–4645 (1997). A crosslinked network ofpoly-methylmethacrylate has been synthesized using an ethylene glycoldi(1-methacryloyoxy)ethyl ether crosslinker that is breakable andpH-responsive, described by Ruckenstein E. et al., Macromolecules. 32,3979–3983 (1999). But networks synthesized with this crosslinker onlydegrade under strong acidic conditions, such as pH 2.0 and below. Thehydrolysis of this type of linkage is not acid catalyzed at mildlyacidic levels present in biological applications. Thus, there is a needfor hydrogels and microgels that degrade under mildly acidic conditions.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to microgels for application in thedelivery of proteins, vaccines, drugs (such as the anticancer drugscisplatin, paclitaxel or taxitere), and other bioactive materials. Themicrogels comprise crosslinked polymer hydrogels of microparticle size,that contain or incorporate bioactive materials. The hydrogelcompositions are made using an inverse microemulsion technique (aqueousdroplets in an oil or aliphatic phase) that results in microgels of apredetermined size, typically 0.1–10 microns in diameter. A size rangebetween 200 nm and 500 nm is optimal for phagocytosis by immune cells.

The microgels of the current invention release their contents inresponse to the mild acidic conditions found in lysosomes, tumors,inflammatory tissues and the phagolysosomes of antigen presenting cells.The present crosslinkers will hydrolyze at a preferred pH range of 4.5to 6.8, more preferably pH 5 to 6. Prefereably, they will completelyhydrolyze within 24 hours at pH 5. The current invention alsospecifically describes hydrogels and microgels synthesized with abisacryloyl acetal crosslinker that hydrolyzes under acidic conditions,such as in the phagolysosome, and releases the encapsulated contents ofthe hydrogel or microgel after entering a cell.

The present invention provides a method of preparing a microgelcomposition for delivering a bioactive material to a cell, comprisingthe steps of (a) preparing a mixture which contains the bioactivematerial, a polymerizable group (i.e. the monomer to be polymerized),and a crosslinking group in an inverse emulsion where the aqueous phasecontains the polymerizable group and the cross linking agent, and thealiphatic phase contains the bioactive material; (b) sonicating themixture to achieve a pre-determined particle size, generally 0.1 to 100microns, as determined by the time of sonication; (c) polymerizing thepolymerizable group and the crosslinker in the presence of the bioactivematerial; and (d) recovering the resulting microgel preparation havingbioactive material bound inside. Polymerization is carried out accordingto known reaction parameters, i.e. with a known initiator (such aspotassium peroxodisulfate), and an optional catalyst such as TEMED.Alternatively the microgels are prepared and the bioactive material canbe adsorbed onto the surface of the microgels, or reacted to the surfaceof the microgels.

The present invention thus provides an acid hydrolyzable microgelcomposition for delivering a bioactive molecule, comprising: an acidhydrolyzable microgel composition for delivering a bioactive molecule,comprising: a polymer backbone, which may be acrylic, dextran or othercrosslinkable polymer, linked by a crosslinker; the crosslinker havingthe formula R²CH(OR¹)₂, wherein R¹ is a crosslinkable, acid hydrolyzablelinkage selected from one of compounds (a)–(f) of FIG. 2, R² is Ar—Xwhere X is a water solubilizing group selected from hydrogen, methoxy,—O—(CH₂—CH₂—O)_(n)—CH₃ wherein n is from 1 to 10,—O—CH₂—CH₂—O—C(O)—O—Ph—NO₂ and —O—CH₂—CH₂—CH₂—NH—CO-(dextranpolysaccharide), said dextran polysaccharide having a molecular weightfrom 300 to 100,000 daltons, preferably 300–10,000 daltons; and Ar is ahomocyclic aromatic radical, whether or not fused, having 6 to 12 carbonatoms optionally substituted with one to three substituents; a particlesize between 0.1–10 microns and cross linkages between 1 and 20 molepercent, sufficient to physically trap the bioactive molecule within themicrogel. In accordance with convention, “Ph” represents phenyl.

In cases where dextran is used, dextran may serve as a watersolubilizing group (part of the cross linker) and the polymer backboneitself. In these cases, the crosslinker of the formula R²CH(OR¹)₂containing dextran is reacted with itself in the presence of a radicalsource (and the bioactive material) to form the microgel composition. Inthis case, R is Ar—X where X is an alkyl dextran, wherein said dextranhas a molecular weight from 300 to 100,000 daltons, preferably300–10,000 daltons and an alkyl group links the aryl group to the linkeddextran. In this embodiment, the R¹ groups crosslink to other R¹ groups.In contradistinction, in the acrylic polymer embodiments, thecrosslinker acts through side groups (e.g. carboxyl) on the polymerbackbone formed by the polymerizing units. The particle prepared asdescribed above will have cross linkages between 1 and 20 mole percent,based on the ratio of cross linker added. This degree of crosslinking issufficient to physically trap the bioactive molecule within themicrogel.

Where dextran is used, the present synthetic strategy involves thepreparation of an “activated dextran,” (e.g. compound 838 in FIG. 8B andcompound 914 in FIG. 9B) which is attached to a crosslinker precursorthrough the R¹ group as represented in FIG. 2. The “activated dextran”has a fraction of the glucose moieties (approximately one in six)modified at the 4 ring position to carry a reactive group such as anamine group or a nitrate group for coupling to the crosslinker precursorR group.

The above compositions preferably contain a bioactive material which isselected from the group consisting of consisting of polysaccharides,proteins, DNA and RNA. The DNA may be unmethylated (e.g. bacterialplasmid) DNA, which evokes an immune response in mammals. The biologicalmaterial may also be a protein or other chemical antigen that isdelivered to the lysosome of an immune cell for antigen presentation.This provides an effective vaccine.

The composition may also be designed so that R¹ is (a) or (b) in FIG. 2and R² is such that Ar is phenyl and X is methoxy. This composition mayfurther comprise a bisacryloyl acetal crosslinker for use incrosslinking acrylic polymers. A bisacryloyl acetal crosslinker willhave the formula: R²CH(OR¹)₂, wherein R¹ is selected from one ofcompound (a)–(d) of FIG. 2 and R² is Ar—X where X is—O—(CH₂—CH₂—O)_(n)—CH₃ wherein n is from 1 to 10, and aryl is ahomocyclic aromatic radical, whether or not fused, having 6 to 12 carbonatoms optionally substituted with one to three substituents.

The present invention also comprises methods of preparing the compoundsdescribed herein. The methods may be particularly adapted to thesynthesis of the triglyme or tetraglyme crosslinkers shown in FIGS. 6and 7 respectively. These methods comprise the steps of (a) preparing a1-chloro oxyalkane according to the desired n; (b) reacting the compoundof step (a) with with hydroxybenzaldehyde to produce anoxyalkane-benzaldehyde; (c) converting the compound of step (c) to anacetyl with a 2,2,2-trifluoracetamide; and (d) cleaving said2,2,2-trifluoro groups and reacting the intermediate with acryloylchloride.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the application of the gelmaterials in microparticle form being applied to an antigen presentingcell.

FIG. 2 is a table showing various compositions for the present crosslinkers using the generic formula R²—CH(—OR¹)₂.

FIG. 3 is a synthetic scheme showing the manufacture of a microgelhaving a polymer, a cross linker, and a bioactive material, and thesubsequent dissolution of the particle.

FIG. 4 is a schematic showing the synthesis of bisacrylamidemethoxybenzaldehyde acetal crosslinker.

FIG. 5 is a schematic showing the synthesis of autocatalytic bisacryloylacetal crosslinkers. Synthetic routes (5A) to these crosslinkers andtheir degradation under acidic conditions (5B) are shown.

FIG. 6 is a schematic showing the synthesis of bisacrylamide triethyleneglycol (triglyme) acetal crosslinker.

FIG. 7 is a schematic showing the synthesis of bisacrylamidetetraethylene glycol (tetraglyme) acetal crosslinker.

FIG. 8 is a schematic showing the synthesis of a bisacrylamidenitrochloroformate acetal crosslinker 836 and dextran microgels. FIG. 8Ashows the synthesis of the bisacrylamide nitrochloroformate acetalcrosslinker 836. FIG. 8B shows the conjugation of the crosslinker 836with activated dextran to yield a dextran acetal crosslinker 840. FIG.8C shows synthesis of dextran microgels using crosslinker 840.

FIG. 9 is a schematic showing the synthesis of bisacrylamide dextranacetal crosslinker 920 and dextran microgels. FIG. 9A shows thesynthesis of the intermediate compound bisacrylamide amine acetal 910.FIG. 9B shows the conjugation of the amine acetal 910 with activateddextran to yield a bistrifluoroacetamide dextran acetal 916. FIG. 9Cshows the modification of the intermediate acetal 916 to make thebisacrylamide dextran acetal crosslinker 920. FIG. 9D shows synthesis ofdextran microgels using crosslinker 920.

FIG. 10 is a schematic showing dextran microgels upon acid hydrolysisand biodegredation.

FIG. 11 is a schematic showing the synthesis to activate mannose toincorporate immunostimulatory groups such as mannose into microgels toincrease CTL activation.

DESCRIPTION OF THE PREFFERRED EMBODIMENT Definitions

The term “hydrogel” herein refers to a three dimensional macromolecularnetwork in water that is formed from a cross-linked polymer.

The terms “microgel”, “microgel particles” and “microparticle” hereinrefers to a three-dimensional hydrogel particle that is 0.1–10 μm indiameter.

The term “bioactive particle” herein refers to a composition having aphysiological effect in a cell, particularly a protein antigen, DNA, andenzyme or other organic molecule.

The term “acetal” herein refers to a diether in which both ether oxygensare bound to the same carbon.

The term “acryl” or “acryloyl” herein refers to the general structure(CH₂═CH—CO—).

The term “bisacryloyl acetal” herein is used to refer to an acetal withtwo acryl groups, one on each end of the acetal linkage, having thegeneral structure of R²CH(—OR¹)₂, wherein R¹ terminates in the structureC(O)CH═CH₂, as shown in FIGS. 2( a), (b), (c), and (d).

The term “crosslinker” herein refers to a molecule with two or morefunctional groups that can form a three-dimensional network when reactedwith the appropriate co-monomers.

The term “polymerizable group” herein refers to monomers whichpolymerize upon introduction of an initiator or radical source.

The term “aryl” herein refers to a homocyclic aromatic radical, whetheror not fused, having 6 to 12 carbon atoms optionally substituted withone to three substituents, wherein said substituents are preferably N orS.

The term “inverse emulsion” herein refers to an emulsion having anaqueous phase and an oil phase, wherein the continuous phase is the oilphase and the water-soluble droplets are dispersed in a continuous phaseof oil.

The term “acrylic polymer” herein refers to a polymer made frompolymerizing units (monomers) that yield a polymer having a crosslinkable side chain, represented as follows:

wherein said monomers are acrylic acid, acrylamide, or various monomersand mixtures thereof having hydrogen substitutions such as NH₂ at the CHgroup.

The term “loading efficiency” herein refers to the percentage of thestarting amount of bioactive material that is encapsulated per milligramof the microgels (μg material/mg microgel) on average, based on thestarting bioactive material/monomers ratio.

The term “alkyl-dextran” herein refers to a dextran polymer having aglycosidic linkage from a dextran through an alkyl spacer group whereinC is less than 100 and optionally substituted with amide bonds andpolyethylene glycol (—O—(CH₂—CH₂—O)_(n)— where n is less than 10.

The term “lower alkane” herein refers to an aliphatic linear or branchedchain or cyclic compound of the formula C_(n)—H_(2n+2), where n isbetween 2 and 20, such as hexane, octane, nonane or the like.

The term “mole percent herein refers, when used in connection withdegree of crosslinking, that degree of cross linking as measured by themoles of crosslinker divided by the total moles of crosslinker andpolymerizable groups.

Introduction

FIG. 1 shows a schematic diagram illustrating the overall compositionand use of the present microgels. The microgels 10 of the currentinvention are loaded with bioactive material 40 including but notlimited to, antigens, proteins, polynucleotides, polypeptides, and otherbioactive material 50. The microgels 10 of the current invention shouldbe synthesized with polymerizable groups 30 and a bisacryloyl acetalcrosslinker 20 that hydrolyzes under acidic conditions and releases theencapsulated contents 40 in response to mildly acidic conditions. Themild acidic conditions found in the body such as in tumors, inflammatorytissues and in cellular compartments such as lysosomes andphagolysosomes 50 of antigen presenting cells 60 should cause the acetalgroup of the bisacryloyl acetal crosslinker 20 to be hydrolysed therebydegrading the microgel and releasing its contents.

In a preferred embodiment, the microgels are delivered to antigenpresenting cells and then phagocytosed and trafficked to the lysosome orphagolysosome of the cells. The mild acidic conditions found inlysosomes and phagolysosomes of APCs should cause the acetal group ofthe bisacryloyl acetal crosslinker to be hydrolysed thereby degradingthe microgels. This acid hydrolysis of the bisacryloyl acetalcrosslinker increases the pore size of the microgels allowing theentrapped bioactive material to diffuse out. This swelling of themicrogels increases the osmotic pressure inside the cellular compartmentwhich causes the cellular compartment to burst, thus releasing thebioactive material into the cytoplasm where it is exposed to the MHCIprotein. The MHCI protein should then display the bioactive material onthe cell surface of the antigen presenting cell and activate cytotoxic Tlymphocytes (CTL) which can then recognize virus, infected cells thatdisplay the bioactive material, thus targeting CD⁺8 immune response.

A. The Bisacryloyl Acetal Crosslinker

1. Designing the Crosslinker

In a general embodiment, the bisacryloyl acetal crosslinker of theinvention is stable to basic conditions but hydrolyzes rapidly in acidicenvironments. Referring now to FIG. 2, a generic formula for thebisacryloyl acetal crosslinker (labeled as Compound I) is a generalstructure of R²CH(—OR¹)₂, where R¹ contains an acryl polymerizing group,and R² is a water solubilizing group. Acid degradable microgels can thenbe synthesized by copolymerizing this crosslinker with polymerizablegroups in the presence of a bioactive material.

In general, the design of the bisacryloyl acetal crosslinker alsoreflects such factors as ease of synthesis, solubility, commerciallyavailable reagents, the type of hydrogel or microgel particle desired,the loading efficiency, dispersion of the particles, toxicity and thehydrolysis rates of the acetal linkage.

2. Appropriate Acryloyl Groups (R¹)

A key factor in choosing acryloyl groups is the chosen synthesisstrategy of the crosslinker. Appropriate acryloyl groups (or acrylpolymerizing group) that can be used for the bisacryloyl acetalcrosslinker of this invention include but are not limited to,ethylacrylamides (a), methylacrylamides (b), acrylates (c), acrylamides(d), trifluoro-, tribromo-, trichloro- and triiodo-acetamides (e) andethylamines (f). In a preferred embodiment, the acryloyl group (R¹) isan acrylamide, a substituted acrylamide, such as a methyl or ethylacrylamide, acrylates or acrolein groups, substituted amides, andsubstituted vinyl groups.

3. Acid Degradable Linkages and Water Solubilizing Groups (R²)

The present crosslinkers that are stable at higher than pH 7.0 buthydrolyze at a pH preferably about 5. The bisacryloyl acetal crosslinkershould have an aqueous solubility of greater than 50 mg/ml. Thissolubility is important to insure that the microgels will bepolymerizable under inverse microemulsion conditions.

Appropriate water solubilizing groups to use for R² to create the acetallinkage are compounds that contain polar groups and/or good leavinggroups. Water solubilizing groups include but are not limited to, an,aryl, alkylaryl, alkoxy, aroxy or diaryl group, benzaldehyde ormethoxybenzaldehyde.

The acid degradable crosslinker of the invention has advantages overother types of linkages in the prior art. In contrast, the crosslinkerof the invention disclosed herein hydrolyzes on the timescale in whichendosomes mature into lysosomes, in contrast to the extremely slowhydrolysis rates of cyclic ketals.

4. Water Solubilizing Groups (R²) with Functional Groups

In a preferred embodiment, the crosslinker in this invention uses anaromatic acetal as the acid degradable linkage. The aromatic acetal hastwo important advantages over ketals, (i) their hydrolysis rates can becontrolled by adding substituent water solubilizing groups in their paraposition, and (ii) the aromatic portion of the acetal can also act as afunctional group for conjugation to biodegradable polymerizable groups,such as dextrans.

Referring now to FIG. 5A, in a preferred embodiment, an autocatalyticbisacryloyl acetal crosslinker can be used to design novel types ofmicrogels in which an amplification of the rate of release of bioactivematerial is provided by each cleavage step. A strategy for using anautocatalytic bisacryloyl acetal crosslinker involves the condensationof one molecule of a carbonyl compound, such as a benzaldehyde, with twomolecules of a carboxylic acid or derivative thereof. For example, FIG.5A shows an bisacrylic benzaldehyde acetal crosslinker 530 obtained byincorporation of two acrylic acid moieties in an acetal like structurewith benzaldehyde.

As shown in FIG. 5B, hydrolysis of the autocatalytic crosslinker 530under mild acidic conditions proceeds with release of two molecules ofacrylic acid. Such release contributes to increase the acidity of themedium, thereby accelerating further hydrolysis. Amplification isdesirable as it may contribute to faster release of the encapsulatedbioactive material possibly enabling release from the normally lessacidic endosomes or other compartments of cells. Preparation ofmicrogels using such an autocatalytic crosslinker can also be carriedout using inverse emulsion polymerization with suitable co-monomersincluding but not limited to, hydroxymethyl methacrylate or acrylamide.

In a more preferred embodiment, R² of the bisacryloyl acetal crosslinkeris a benzaldehyde acetal with a para water solubilizing group to ensurethat microgels made with the crosslinker hydrolyze rapidly afterexperiencing the pH 5.0 environment of acidic cellular compartments. Thebisacryloyl acetal crosslinker becomes acid degradable because it hastwo ethers connected to the same carbon. The para water solubilizinggroup is electron donating and causes rapid hydrolysis. The watersolubilizing groups can be positioned in the ortho- and meta-positionsof the benzaldehyde, however, the ease of synthesis and availability ofsuitable starting reagents may make these crosslinkers more difficult tosynthesize.

In preferred embodiments, water solubilizing groups include but are notlimited to, an alkoxy, aroxy or diaryl group, benzaldehyde ormethoxybenzaldehyde, having an ortho or para functional group such astriethylene glycol (triglyme), tetraethylene glycol (tetraglyme),polyethylene glycol, nitrophenylcarbonate, dextrans, saccharides,sugars, and other carbohydrates, and combinations thereof. The size ofthe functional group should preferably have a molecular weight of lessthan 100,000. Linker groups including such groups as(—O—CH₂—CH₂—NH—C(O)—) or (—O—CH₂—CH₂—O—C(O)—O—) may be used to linkthese functional groups to the benzaldehyde acetal and aid in theaddition and synthesis of these crosslinkers.

In specific embodiments, the bisacryloyl acetal crosslinker has abenzaldehyde acetal with a para functional group selected from the groupconsisting of: hydrogen, aldehyde, dimethyl amine, methoxy, triethyleneglycol, tetraethylene glycol, polyethylene glycol, andnitrophenylcarbonate.

5. Hydrolysis of the Crosslinker for Hydrogels and Microgels

The bisacryloyl acetal crosslinker can be hydrolyzed to release thecontents entrapped in the microgels of the invention in a pH dependentmanner. In the preferred embodiment the bisacryloyl acetal crosslinkershould preferably have a half-life at pH 5.0 of 5 minutes to 24 hours at37° C., but a longer half life at pH 7.4 of at least 24 hours to 250days.

In some embodiments, it may be useful for the crosslinker to have ahalf-life at pH 5.0, 37° C. of about 24 hours, and a half-life at pH7.4, 37° C. of about 250 days, in order to facilitate slow release ofbioactive materials. In other embodiments, it is contemplated that thehalf-life of the crosslinker at pH 5.0, 37° C. preferably be 5–30minutes, and even more preferably be less than 5 minutes and a half-lifeat pH 7.4, 37° C. of about 24 hours in order to quickly release thebioactive materials.

The acceleration of the hydrolysis kinetics of a bisacryloyl acetalcrosslinker from pH 7.4 to pH 5.0 is expected because the hydrolysis ofthe acetal is proportional to the hydronium ion concentration, whichshould increase between pH 7.4 and pH 5.0. The kinetics of acetalhydrolysis can be easily manipulated by introducing the appropriateelectron withdrawing or donating groups and therefore it is possible toengineer acetal crosslinked hydrogels that have hydrolysis ratestailor-made for a given application.

The hydrolysis kinetics of the bisacryloyl acetal crosslinker changesafter its incorporation into the microgels. This change in thehydrolysis kinetics of the crosslinker is potentially due to twofactors, (1) the steric effects of tethering the acetal moiety of thecrosslinker into the particle, which in effect generates a cyclic acetal(cyclic acetals hydrolyze 10–100 times slower than straight chainacetals because of steric reasons) and (2) the diffusion of thehydronium ion into the microgels.

A kinetic factor that may be taken into account when designing thebisacryloyl acetal crosslinker is the crosslinker's speed of hydrolysisin solution. In an embodiment where the goal is to hydrolyze thecrosslinker and rapidly release the bioactive material, the bisacryloylacetal crosslinker should preferably hydrolyze within 5–30 minutes at pH5.0 at 37° C. This timescale is chosen because it is approximately theamount of time taken for a phagocytosed gel particle to be trafficked tocellular compartments such as lysosomes. In a preferred embodiment,these particles will degrade rapidly in the lysosome and cause lysosomaldisruption. Having a particle that degrades too slowly will increase itsresidence time in the lysosome and allow the lysosomal enzymes increasedchances of hydrolyzing the bioactive material before reaching thecytoplasm through lysosomal disruption. Therefore, in a preferredembodiment, the crosslinker should hydrolyze fairly rapidly at apreferred range of pH 7.4 to 4.5 and even more preferably between pH 6.8to 4.5.

6. Synthesis of the Crosslinker

In a general embodiment, the bisacryloyl acetal crosslinker has thegeneral structure of R²CH(—OR¹)₂, which can be made by firstsynthesizing the R² water solubilizing group.

In one embodiment, the strategy for synthesis is reacting the watersolubilizing group with an amino alcohol that has its amine groupprotected with an acid stable protecting group, such as atrifluoroacetamide, to form an acetal. The protected amines can then bedeprotected and reacted with acryloyl chloride to generate thebisacrylamide acetal crosslinker. If an alcohol is used having no aminogroup, it can be reacted with an acryloyl chloride to make an acrylate.However, use of an acrylate may raise some solubility issues asacrylates tend to be 100-fold less soluble in the aqueous phase asacrylamides.

In a second embodiment, the strategy for synthesis involves thecondensation of one molecule of the water solubilizing group with twomolecules of a carboxylic acid or derivative thereof to generate anautocatalytic bisacryloyl acetal crosslinker.

In a preferred embodiment, the crosslinker is selected from the groupconsisting of: bisacrylamide methoxybenzaldehyde acetal crosslinker 110(as shown in FIG. 4), bisacrylic benzaldehyde acetal crosslinker 530 (asshown in FIG. 5A), bisacrylamide triglyme acetal crosslinker 606 (asshown in FIG. 6), bisacrylamide tetraglyme acetal crosslinker 710 (asshown in FIG. 7), bisacrylamide nitrochloroformate acetal crosslinker836 (as shown in FIG. 8A) and bisacrylamide dextran acetal crosslinker920 (as shown in FIG. 9C).

Acid degradable microgels can then be synthesized by copolymerizing thisbisacryloyl acetal crosslinker with polymerizable groups in the presenceof a bioactive material.

B. Microgel Particles for the Delivery of Bioactive Materials

Microgels made with the bisacryloyl acetal crosslinker shouldefficiently entrap bioactive material with a comparable loadingefficiency. In a preferred embodiment, the microgel particle size mayvary between 0.1–10 μm and exhibit a loading efficiency of at least 40%bioactive material encapsulation, more preferably at least 50% loadingefficiency and even more preferably at least 54% loading efficiency.

1. Appropriate Polymerizable Groups for Gel Particles

Appropriate polymerizable groups that can be used for the microgels ofthis invention include acrylic polymers such as, acrylamides,methacrylamides, methacrylates, and acrylates.

In more preferred embodiments, the polymerizable groups are acrylamidesor methacrylamides.

Appropriate biocompatible polymerizable groups that can be used for themicrogels of this invention include biocompatible polymers including butnot limited to, dextrans, saccharides, mannoses, sugars, carbohydrates,nucleic acids, oligonucleotides, amino acids, polypeptides, lipids andcombinations thereof.

In a more preferred embodiment, the biocompatible polymerizable group isdextrans up to 100,000 MW, more preferably up to 10,000 MW. In thisembodiment, the biocompatible polymerizable group is conjugated to thefunctional group X of R² of the Compound I bisacryloyl acetalcrosslinker before synthesis of the microgels.

2. Bioactive Materials

In a preferred embodiment, the invention contemplates entrapping suchbioactive materials including but not limited to, nucleotides,polynucleotides, ribonucleotides, amino acids, peptides, proteins,antigens, plasmid DNA, growth factors and hormones, interleukins,immunostimulatory agents, drugs, vaccines, neuromodulatory agents suchas neurotransmitters, stimulatory and adrenergic agents, enzymes,proteases, anticancer and antitumor agents, imaging agents, diagnosticagents, antiviral agents and antibacterial agents.

In specific preferred embodiments, the bioactive material is selectedfrom the group consisting of: nucleotides, polynucleotides, proteins,immunostimulatory agents, vaccines, antigens, anti-viral agents, proteinantigens, anticancer agents and antitumor agents.

These bioactive materials can be conjugated with a carrier molecule orthey can be conjugated to a polymerizable group and copolymerized intothe microgels. The linkage between the polymerizable group and thebioactive molecule can be designed to be cleaved under variousphysiological conditions. The bioactive material can also be adsorbedonto the surface of the microgels, or reacted to the surface of themicrogels.

3. Synthesis of Microgels

In general, microgels can be synthesized by inverse microemulsionpolymerization according to the procedure described by Kriwet, B.;Walter, E.; Kissel, T.; J. Control Release, 1998, (56), 149–158, whichdescribes synthesis of bioadhesive poly(acrylic acid) nano- andmicroparticles using an inverse emulsion polymerization method for theentrapment of hydrophilic drug candidates. A key issue in the synthesisof microgels by inverse emulsion polymerization is the aqueoussolubility of the acryloyl groups and polymerizable groups. Thesolubility of both the acryloyl groups and polymerizable groups are veryimportant as all of the polymerizable components in an inverse emulsionpolymerization must be sufficiently water soluble.

During the inverse microemulsion polymerization, a small amount of wateris dispersed into an organic phase and stabilized by surfactants.Sonication before polymerization for about 5 minutes will insure thecorrect particle size, which will cover a range of sizes, within therange of about 100 nm–10 μm, preferably 100 nm–5 μm. The polymerizablegroups and the bisacryloyl acetal crosslinker are then polymerized inthe aqueous phase in the presence of the bioactive material and aninitiator molecule or radical source. Since polymerization is initiatedand contained within water droplets, mainly spherical crosslinkedmicrogel particles containing entrapped bioactive material are produced.To adjust particle size, either longer sonication time or largersurfactant concentration will decrease the microgel particle size.

Several different emulsion polymerization procedures were attemptedusing different organic phases and surfactant blends. Inversepolymerizations with toluene/chloroform as the continous phase andpluronic F-68 as the surfactant were unsuccessful. However, inversepolymerizations with hexane as the continous phase and SPAN™ 80(sorbitan monooleate), TWEEN™ 80 (polyethyleneglycol-sorbitanmonooleate), dioctyl sulfosuccinate (AOT) and Brij 30 (Polyoxyethylene(4) Lauryl ether) (all from Sigma Aldrich, St. Louis, Mo.) assurfactants are successful in producing microgels.

That the bisacryloyl acetal crosslinker exhibits improved performance informing microgel particles in hexane/water versuschloroform-toluene/water is potentially explained by the lowersolubility of the crosslinker in hexane versus chloroform-toluene. Forexample, the bisacryloyl acetal crosslinker has water/hexane partitionratio of 10,000:1. In contrast, the water/toluene-chloroform partitionratio is only 1:1, suggesting that in the water/chloroform-toluenepolymerizations, a large fraction of the crosslinker is lost in theorganic phase. Thus, the organic phase is most preferably hexane and thesurfactants used are preferably TWEEN™ 80, SPAN™ 80, dioctyl sulfosuccinate (AOT) and Brij or combinations thereof. More preferably thesurfactant used is a 1:3 ratio of TWEEN™ 80/SPAN™ 80. Furthermore, it isimportant that the surfactants used for synthesis be neutral and FDAapproved for human use. Neutral, biocompatible surfactants are preferredfor the synthesis of bioactive material-loaded microgel particlesbecause of their reduced interactions with proteins and lower toxicity.

4. Loading and Loading Efficiency of Entrapped Bioactive Materials

The bisacryloyl acetal crosslinker can affect that loading efficiencyand the amount of bioactive material entrapped in the microgels of theinvention. Loading efficiency is the amount of bioactive material thatis entrapped within the gel particles as compared to the total startingamount of bioactive material placed in the polymerization reaction. Ingeneral, the loading efficiency of the microgel particles of theinvention should not appreciably change with the crosslinking ratio,however, the water solubilizing groups can change the loading andencapsulation efficiencies.

The loading efficiency is different from the amount of proteinencapsulated in a single particle. It is estimated that approximately 1million protein molecules of about 50 kD size can be encapsulated in amicrogel. This number comes from assuming the microgel has a density of1, a radius of 0.5 microns, and are composed of 10% protein by weight.

In a general embodiment, the microgel particles of the invention shouldhave at least a 20% loading efficiency, more preferably 40% loadingefficiency, even more preferably at least 50% loading efficiency, andmost preferably more than 55% loading efficiency.

In a preferred embodiment, wherein the bioactive material loaded is DNAmaterial, the loadings and efficiencies of the microgel particles shouldbe comparable to other microparticle systems which have efficienciespurported to be about 1–2 μg DNA/mg polymer for 500 nm PLGA particles.(See Garcia del Barrio, G.; Novo, F. J.; Irache, J. M. Journal ofControlled Release (2003), 86(1), 123–130). It is estimated that about3,000–7,000 molecules of DNA can be encapsulated within a singlemicrogel of the present invention, if the DNA encapsulated was 6,000 bp,which has a MW of about 4 million daltons. The loading efficiencies forthe amount of DNA material entrapped in microgel particles of thepreferred embodiment should preferably be at least 40%, more preferablyat least 50% and even more preferably at least 54%.

In a preferred embodiment, wherein the bioactive material loaded isprotein, the loading efficiencies for the amount of protein entrapped inmicrogel particles of the preferred embodiment should be at least 20%,preferably at least 40%, more preferably around 50%.

The loading efficiency and the amount of bioactive material entrapped isan important aspect in light of such factors as the amount of bioactivematerial needed to be delivered to the target for an effective dose andthe amount of available bioactive material. A major drawback in previoustherapeutics and vaccines is there is often difficulty in obtaininglarge enough amounts of the therapeutic composition of bioactivematerial for production. Therefore, it is a goal of the invention tomake microgels with high loading efficiencies so as to lower thestarting amount of bioactive material required at the beginning ofpolymerization.

D. Release of Entrapped Bioactive Materials

The release of bioactive materials from the loaded gel particles madewith the bisacryloyl acetal crosslinker can be first measured at variousmild pHs at 37° C., in aqueous solutions. In a preferred embodiment, thecrosslinker should hydrolyze within 5 minutes to 24 hours at pH 5.0 andhave a much slower hydrolysis rate at pH 7.4.

In a preferred embodiment, these microgels will degrade rapidly in thelysosomes and cause lysosomal disruption. Therefore, in a preferredembodiment, at a more acidic pH 5.0, encapsulated bioactive materialsshould preferably be 80% released from the microgel particles within 6hours, preferably completely released from the microgel particles within12 hours, more preferably within 8 hours, and even more preferablywithin 6 hours. At pH 7.4, the release of entrapped bioactive materials40 should be significantly slower, taking up to 150 hours for themicrogel particles to completely release their contents.

The molar ratio between the concentration of polymerizable groups andthe bisacryloyl acetal crosslinker affects the rate of bioactivematerial released from the gel particles. It is preferred that themicrogels of the present invention have 1–20 mole percent crosslinking,between 1%–12.8% crosslinking, and most preferred between 1%–3%crosslinking, but sufficient to physically trap the bioactive moleculewithin the microgel.

For example, a molar ratio of 9:1 acrylamide/bisacryloyl triglyme-acetalcrosslinker (1.6% crosslinking) results in a linear release of bioactivematerial, with nearly 80% released at 300 minutes. A ratio of 4:1 ofacrylamide to bisacryloyl triglyme-acetal crosslinker (3.5%crosslinking) results in a steeper initial increase in release ofbioactive material with a slower increase from 100–200 minutes and ansecond steep increase from 200–300 minutes. A small molar ratio of 1:1acrylamide/bisacryloyl triglyme-acetal crosslinker (12.8% crosslinking)results in a very slow release of 20% of the bioactive material in thefirst 175 minutes and a steep release of bioactive material.

Thus, depending on the desired rate of release of bioactive material,the amount of crosslinker can be increased or decreased. In general,1–3% crosslinking may be preferred, not only for its steady linearrelease of bioactive material, but also because of the crosslinker'seffects upon other factors such as toxicity, loading efficiency, andamount of T-cell activation. But it is contemplated that in someembodiments, increased amounts of crosslinker may be more preferred,such as in a case for example, where the crosslinker size is small.

E. Antigen Presentation and T-cell Activation

The microgels release their bioactive material payload into thecytoplasm of cells upon lysosomal disruption. Higher loading capacity ofthe gel particles may also lead to greater antigen presentation of theencapsulated bioactive material.

In the antigen presentation assay described by Sanderson, S.; Shastri,N. in Inter. Immun. 1994, 6, 369–376, the β-galactosidase activity ofB3Z T cells is measured. Antigen presenting cells display the peptidehaving the sequence, SINFEKL, upon phagocytosis of ovalbumin. Thesecells were engineered to transcribe β-galactosidase when in the presenceof antigen presenting cells displaying the SINFEKL peptide.β-galactosidase then liberates chlorophenol red from the chlorophenolred βgalactoside that is present in solution. Absorbance of chlorophenolred is measured by UV absorbance at 595 nm. Therefore this assay can beused as a measurement of the amount of bioactive material delivered intothe cytoplasm of cells by the microgel particles of the invention bymeasuring the amount of liberated chlorophenol red by UV absorbance at595 nm.

A proper control would be to compare the amount presented by the gelparticles 10 of the invention when incubated with the SINFEKL peptidewhich is directly displayed on the antigen presenting cells and notdelivered to the cytoplasm of the cells first. A maximum absorbance of0.25 should be observed, which results in 100% T-cell activation. In apreferred embodiment, the bioactive loading capacity and efficiencyshould lead to an absorbance of preferably at least 0.15, morepreferably 0.2, and most preferably more than 0.25, using the antigenpresentation assay described by Sanderson, S.; Shastri, N. in Inter.Immun. 1994, 6, 369–376.

A preferred basic minimal level of antigen presentation that theparticles should effectuate is about 50% T-cell activation as theminimum level of T-cell activation. Efficient microgels should needapproximately 500 micropaticles per antigen presenting cell. The levelbeyond which the starting amount of bioactive material and micrgelscompared to the amount of antigen presentation is inefficient andunpreferred is considered about 5 mg/ml of microgels to generate a 100%T cell activation. This level is inefficient and unpreferred because ofthe potential toxicity involved with the delivery vehicles.

F. Toxicity of Gel Particles and Degraded Gel Particles

Use of this invention in human and mammalian therapeutics brings upissues of the toxicity of these microgels. As the amount of crosslinkerand polymerizable groups increases, the viability of cells decreases.The viability of cells can be measured by the ability of mitochondria inmetabolically active cells to reduce yellow tetrazolium salt (MTT) toform insoluble purpose formazan crystals.

In a preferred embodiment, the target antigen presenting cells shouldpreferably exhibit at least 50% viability after 24 hours of incubationwith the gel particles of the invention, more preferably at least 70%viability after 24 hours, even more preferably at least 80% viabilityand most preferably more than 90% viability after 24 hours according tothe MTT assay as described above and in Example 14.

Polymers with high MW are not easily excreted from body, thereforeanother aspect of the invention is to make microgel particles thateasily and safely excreted by the body after being degraded in theacidic cellular compartment. In general it is preferred that themicrogel particles degrade into linear polymer chains are 100,000daltons or less. Thus, dextran microgels are one preferred embodimentbecause dextrans degrade into chains of 10,000 daltons or less.

Another factor in the size of the degraded particles is the amount ofcrosslinker used. As described in Example 16, lower amounts ofcrosslinker used result in smaller linear polymer chains, thereforemaking excretion of the degraded particles more likely. However, theinability of cells to secrete larger polymer chains may not beproblematic as the amount of particles to which the cells are exposedare very small. Furthermore, the toxicity studies show that cells arestill viable after long-term exposure to the microgel particles.

G. Applications for Acid Degradable Hydrogels and Microgels

This strategy for the synthesis of acid degradable microgels has manyapplications including the delivery of bioactive materials, includingbut not limited to polynucleotides, polypeptides, proteins, peptides,antibodies, vaccines, antigens, genetic, or therapeutic agents, to thecytoplasm of phagocytic cells.

1. Vaccine Therapeutics

In one embodiment, the microgels of the present invention would haveapplications in vaccine therapeutics and disease prevention. Proteinloaded microgels could be injected as an intramuscular injection to apatient, stimulating phagocytosis by macrophages and antigen presentingcells. After being sequestered in lysosomes, the acid degradable linkageof the bisacryloyl acetal crosslinker would hydrolyze, releasing theprotein antigen, and cause the lysosome to swell and then burst, therebyreleasing the lysosome contents into the cellular cytoplasm. Once theprotein antigen is released into the cytoplasm, MHCI proteins can thenbind the protein antigen and present the protein antigen on the cellmembrane. These cells would then initiate the cytotoxic T lymphocyteimmune response against pathogens from which the protein antigen camefrom.

The gel particles of the invention would be particularly useful incombating infections that need a strong cytotoxic T lymphocyte response,including diseases such as HIV/AIDS and Hepatitis C infections. Examplesof such antigens which can be used as bioactive material and entrappedin the microgels of the present invention, include but are definitelynot limited to, the TAT protein from HIV, the ENV protein from HIV, theHepatitis C Core Protein from the Hepatitis C virus, the prostatic acidphosphatase for prostate cancer and the protein MART-1 for melanoma.

2. Gene Therapy

In a second embodiment, the microgels of the invention would be used forgene therapeutics. Since gene therapy involves the delivery of asequence of DNA to the nucleus of a cell, the microgels of the inventionwould be especially suited for this application. Once a polynucleotideis delivered by the microgels to the cytoplasm, the polynucleotide canundergo translation into a protein. This has the potential, then, tomake proteins that are not normally produced by a cell.

In a preferred embodiment, the bioactive material would be a plasmidthat encodes for a protein or antigenic peptide initially. For example,one would use a plasmid that encodes for a protein that would displayantigens for cancer. These proteins would not be easy to generate inmulti-milligram to gram quantities to be delivered to a patient,therefore using the microgels of the present invention to deliverplasmid DNA encoding these antigens is a preferred alternative. PlasmidDNA, encapsulated in the microgels of the invention and delivered to thecytoplasm of phagocytic cells, has been shown to be active and stillable to transfect cells.

In addition to encoding for a gene, plasmid DNA has the addedcharacteristic of generating an immune response because plasmid DNA isgenerated from bacteria. Bacterial DNA has two major differencescompared with vertebrate DNA: 1) bacterial DNA has a higher frequency ofCG dinucleotides in the sequence ( 1/16 dinucleotides in microbial DNAare CG pairs, but only 25% of that is observed in vertebrate DNA); and2) bacterial DNA is unmethylated as compared to vertabrate DNA which isoften methylated. Vertebrate systems will recognize the plasmid DNA thenas being foreign, and the cell will react as for a bacterial infection.This immune response is manifested in the production of cytokines andinterleukins that then go on to activate T cells, B cells, and othercells, proteins, and cellular machinery involved in the immune response.Example 20 demonstrates that bacterial DNA delivered by microgels indeedincreases the production of interleukins.

3. Directing Patient Immune Response

In a further embodiment, the plasmid DNA used as the bioactive materialwould have an added interleukin sequence. (Egan, Michael A.; Israel,Zimra R. Clinical and Applied Immunology Reviews (2002), 2(4–5),255–287.) Interleukins are secreted peptides or proteins that mediatelocal interactions between white blood cells during immune response (B.Alberts et al, Molecular Biology of the Cell, 4th ed., Garland Science,2002). Different interleukins (e.g. IL-12, IL-2) will direct the type ofimmune response that is generated. IL-6, IL-1, 8, 12, and TNF-α aresecreted by infected macrophages as an immune response and IL-6 servesto activate lymphocytes and increase antibody production. Thedifferentiation of helper T cells into either T_(H)1 or T_(H)2 efffectorcells determines the nature of the response. A T_(H)1 response ischaracterized by a CTL response; a T_(H)2 response is characterized byantibody production.

The addition of the interleukin-2 or 12 (IL-2 or IL-12) gene sequence,and its subsequent translation into an interleukin protein may allow thedirection of the type of patient immune response and amplification ofthe desired CTL response by adding or displaying immunostimulatorygroups on the surface of the microgels. Such immunostimulatory groupsinclude but are not limited mannose, plasmid DNA, oligonucleotides,ligands for the Toll receptors, interleukins and chemokines. T-cellsactivate B-cells to secrete Interleukin-6 (IL-6) to stimulate B cellsinto antibody-secreting cells.

It has been shown by Apostolopoulos, V.; McKenzie, I. F. C. CurrentMolecular Medicine (2001), 1(4), 469–474, that activation of the mannosereceptors on the surface of APCs leads to enhanced CTL activation.

Incorporation of mannose onto the present microgels by copolymerizing amannose polymerizable group with the polymerizable groups and thecrosslinker to make microgels should likely result in increased CTLresponse. The synthesis of activated mannose is shown in FIG. 18.

4. Particle Carriers and Dispersion

In other embodiments, oligonucleotides (approximately 12–75 bases inlength) can be used for immunostimulation as well as for their antisenseactivity. However, because oligonucleotides are too small to remainencapsulated inside the microgels of the invention, they must beconjugated to a polymerizable group and then later released. One way tosolve this problem would be to attach the oligonucleotides to apolymerizable group through an acid degradable linkage similar tocreating a second crosslinker, whereby acid hydrolysis in the acidicconditions will release the oligonucleotide.

Alternatively, the oligonucleotides can be conjugated to a largemacromolecule, such as dextran through an acid degradable linkage whichis then physically entrapped in the microgels.

The microgels of the invention can be suspended or stored in aconventional nontoxic vehicle, which may be solid or liquid, water,saline, or other means which is suitable for maintaining pH,encapsulation of the bioactive material for an extended period of time,sufficient dispersion or dilution of the microgels and the overallviability of the microgels for their intended use.

Preferably the microgel particles of the invention are stored in drystate (vacumm dried) and stored at 4° C. for several months. Themicrogels can be dispersed in buffer and sonicated or vortexed for a fewminutes to resuspend into solution when needed. The microgels should bevortexed or sonicated for a sufficient amount of time to resuspend themicrogels evenly in solution, however, not too long as vortexing andsonication can also damage some proteins and bioactive material. Themicrogel particles are ready for delivery upon visual determination thatthe microgels are sufficiently dispersed in solution. The solutionshould be opaque with no visible aggregates floating.

5. Pharmaceutically Effective Delivery and Dosages

The loaded microgels of the invention can be administered by varioussuitable means to a patient, including but not limited to parenterally,by intramuscular, intravenous, intraperitoneal, or subcutaneousinjection, or by inhalation. The delivery of the microgels to a patientis preferably administered by injection once but does not preclude thenecessity for multiple injections that would be required to illicit thedesired level of immune response.

The amount of microgels needed to deliver a pharmaceutically effectivedosage of the bioactive material to effect the CTL response in a patientwill vary based on such factors including but not limited to, thecrosslinker and polymerizing group chosen, the protein loading capacityand efficiency of the gel particles, the toxicity levels of thebiodegraded particles, the amount and type of bioactive material neededto effect the desired response, the subject's species, age, weight, andcondition, the disease and its severity, the mode of administration, andthe like.

One skilled in the art would be able to determine the pharmaceuticallyeffective dosage. In general, the amount of bioactive material thatcould be administered by the microgels of the invention is from 1 ng tomore than 1 gram quantities.

The examples, methods, procedures, treatments, specific compounds andmolecules contained herein are meant to exemplify and illustrate theinvention and should in no way be seen as limiting the scope of theinvention. Any patents or publications mentioned in this specificationare indicative of levels of those skilled in the art to which the patentpertains and are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference.

EXAMPLE 1 Synthesis and Delivery of Microgels

Referring now to FIG. 3, an exemplary process for the synthesis of amicrogel particle for pH-dependent degradation is shown. A polymerizableacrylic group 100 is added to the bisacrylamide methoxybenzaldehydeacetal crosslinker 110 (which is described in the following example)wherein R¹=(a) of FIG. 2 and R²=aryl-methoxy. The polymerizationreaction is carried out by inverse microemulsion, initiated by aninitiator such as TMEDA and diammonium persulfate and carried out in thepresence of bioactive particle 40, and carried out in the presence ofbioactive particle 40.

At neutral pHs, the bisacrylamide methoxybenzaldehyde acetal crosslinker110 remains intact and the release of entrapped bioactive material 40 issignificantly slower or not at all. Under acidic conditions, the acetalgroup hydrolyses and increases the pore size of microgels made with it,releasing the entrapped bioactive materials 40, and degraded particles,in the form of multiple polymer chains 140 and a small molecule aldehyde130.

EXAMPLE 2 Synthesis of a Bisacrylamide Methoxy Benzaldehyde AcetalCrosslinker

Referring to FIG. 4, the bisacrylamide methoxy benzaldehyde acetalcrosslinker 110 was synthesized in two steps. The first step was acetalformation with hydroxy-trifluoroacetamide and methoxy benzaldehyde usingthe procedure of Roelofsen et al., Recueil, 90, 1141–1152 (1971, whichgave a 70% yield of bistrifluoroacetamide methoxy benzaldehyde acetal410 after chromatography. In the second step, the intermediate was thendeprotected with 6N NaOH and reacted with acryloyl chloride to yield thecrosslinker 110 (FIG. 3) in 40% yield after chromatography. In thecrosslinker 110, R² (according to the generic formula of FIG. 2) wasmethoxy benzaldehyde (methoxy aryl).

EXAMPLE 3 Synthesis of an Autocatalytic Bisacryloyl Acetal Crosslinker

A strategy for synthesis of an autocatalytic bisacryloyl acetalcrosslinker involves the condensation of one molecule of carbonylcompound such as a benzaldehyde with two molecules of a carboxylic acidor derivative thereof. In this case, R¹=(c) of FIG. 2, and R²=aryl-H.

FIG. 5A shows a bisacrylic benzaldehyde acetal crosslinker 530 obtainedby incorporation of two acrylic acid moieties in an acetal likestructure with benzaldehyde. Referring now to FIG. 5A, para methoxybenzaldehyde 510 is reacted with acrylic anhydride 520 in the presenceof sulfuric acid to form the autocatalytic bisacrylic benzaldehydeacetal crosslinker 530. Also shown is an alternative synthesis is toreact dibromo-toluene with sodium acrylate 550 to form the crosslinker530.

In addition to the same acid degradable linkage, hydrolysis of theautocatalytic crosslinker under mild acidic conditions proceeds withrelease of two molecules of acrylic acid. Such release contributes toincrease the acidity of the medium, thereby accelerating furtherhydrolysis. This autocatalytic crosslinker can be used to designmicrogels which rely on a hydrolysis reaction as shown in FIG. 5B inwhich an amplification of the rate of release is provided by eachcleavage step. Such amplification is desirable as it may contribute tofaster release of the encapsulated bioactive material possibly enablingrelease from the normally less acidic endosomes compartments of cells.Referring now to FIG. 5B, the cross linker 530 of the present examplewill yield, upon hydrolysis, 2 molecules of acrylic acid 560 andp-methoxy benzaldehyde 540.

Preparation of microgel particles using this release-amplifiedcrosslinker can also be carried out using inverse emulsionpolymerization with suitable co-monomers such as hydroxymethylmethacrylate or acrylamide.

EXAMPLE 4 Synthesis of Bisacrylamide Triethylene Glycol AcetalCrosslinker

Referring now to FIG. 6, a more hydrophilic acid degradable crosslinker,containing a hydrophilic triethylene glycol (triglyme) moiety is shown.Since the crosslinker of Example 2 is relatively hydrophobic, it washypothesized that decreasing its hydrophobicity would improve itsperformance since inverse emulsion polymerizations are very sensitive tothe hydrophobic/hydrophilic balance of the reactants. This modificationdramatically increased the hydrophilicity of the crosslinker making itcompatible with a variety of inverse emulsion polymerization procedures.

The bisacrylamide triethylene glycol acetal crosslinker (606) ortriglyme crosslinker as shown in FIG. 6 was synthesized in four steps ona multigram scale. The first step involved the preparation of1-chloro-3,6,9-trioxadecane (603) using the procedure of Loth, H. &Ulrich, F. (1998) J. Control Release. 54, 273. Compound 603 was thenused to alkylate hydroxy benzaldehyde (605) and producep-(1,4,7,10-Tetraoxaundecyl)benzaldehyde (604). Compound 603 was chosenas the alkylating agent since it can be easily synthesized on a largescale (100 grams) enabling the preparation of 4 on a 20 gram scale (70%yield), using potassium carbonate as the base and 18-crown-6 as thephase transfer catalyst. Hydroxy-benzaldehyde could also be alkylatedusing 1-tosyl-3,6,9-trioxadecane and 1-bromo-3,6,9-trioxadecane butthese approaches were discontinued because significantly lower yields ofthe desired product were obtained.

Compound 604 was converted to an acetal (605) by reaction withN-(2-hydroxyethyl)-2,2,2-trifluoroacetamide. A potential problem withacetal formations is the separation of the product from residualalcohol. The alcohol is generally used in 4–6 fold molar excess over thealdehyde, and even high yielding reactions leave 2–4 molar equivalentsof the alcohol to be removed. Initial attempts at purifying the acetalproduct 605 from unreacted N-(2-hydroxyethyl)-2,2,2-trifluoroacetamideusing flash chromatography were unsuccessful. However, 605 could bepurified by crystallization from ethyl acetate/hexane, allowing for itssynthesis on a multigram scale. The final bisacrylamide triethyleneglycol acetal crosslinker was obtained by cleaving the trifluoroacetylgroups on 605 in 6 M NaOH/Dioxane followed by reaction of the resultingdiamine with an excess of acryloyl chloride. Final purification of thecorsslinker was achieved by crystallization from ethyl acetate/hexane.

1-Chloro-3,6,9-trioxadecane (603). Spectroscopic data agreed with thosereported in the literature. ³C NMR (CDCl₃): δ 42.16, 58.30, 69.92,69.96, 69.98, 70.70, 71.30. Anal. Calcd. for C₇H₁₅O₃Cl: C, 46.03; H,8.28. Found: C, 45.68; H, 8.39, yield 70%.

p-(1,4,7,10-Tetraoxaundecyl)benzaldehyde (604). Chloride 603 (26 g, 0.14mol, 1.3 equiv) and p-hydroxybenzaldehyde (13 g, 0.11 mol, 1 equiv) weredissolved in dry THF (40 mL). K₂CO₃ (15 g, 0.11 mol, 1 equiv) was addedfollowed by 18-crown-6 (3.0 g, 11 mmol, 0.11 equiv) and KI (0.20 g, 1.2mmol, 0.01 equiv). The reaction mixture was stirred at reflux for 48 h.The resulting mixture was cooled to room temperature, and water (200 mL)was added. The product was extracted with 3×350 mL portions of ethylacetate and the combined organic layers were dried and concentrated. Theoil was loaded onto a silica gel column and eluted with a 1:9 mixture ofethyl acetate/hexane, followed by a ratio of 1:4, 3:7, 4:1 of ethylacetate/hexane and finally washed with ethyl acetate to afford 21 g(70%) of 604 as a clear oil. IR (cm⁻¹): 1697 (s), 1165 (s). ¹H NMR (400,CDCl₃): δ 3.36 (s, 3), 3.54 (t, 2, J=4.6), 3.63–3.68 (m, 4), 3.72–3.75(m, 2), 3.88 (t, 2, J=4.8), 4.20 (t, 2, J=4.8), 7.01 (d, 2, J=8.8), 9.87(s, 1). ¹³C NMR (CDCl₃): δ 58.86, 67.59, 69.29, 70.40, 70.47, 70.71,71.73, 114.71, 129.84, 13.1.76, 163.69, 190.63. Calcd: [M+H]⁺ (C₁₄H₂₁O₅)m/z=269.13889. Found FAB-HRMS: [M+H]⁺ m/z=269.134487. Anal. Calcd. forC₁₄H₂₀O₅: C, 62.67; H, 7.51. Found: C, 62.47; H, 7.74.

N,N′-Bistrifluoroacetyl-di-(2-aminoethoxy)-[4-(1,4,7,10-tetraoxaundecyl)phenyl]methane(605). Aldehyde 604 (3.60 g, 13.4 mmol, 1 equiv) andN-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (15.0 g, 95.5 mmol, 7.1equiv) were dissolved in dry THF (50 mL). p-Toluenesulfonic acid (0.360g, 2.09 mmol, 0.16 equiv) and 5 Å molecular sieves (50 g) were added.The reaction mixture was stirred overnight and was quenched withtriethylamine (10 mL, 72 mmol, 5.3 equiv). The reaction mixture wasfiltered to remove the molecular sieves with a buchner funnel. A 150 mLportion of water was added to the filtrate and was then extracted withfour 150 mL portions of ethyl acetate. The ethyl acetate was evaporatedand the product was recrystallized twice from ethyl acetate/hexane,recovering 4.03 grams of 605 (60%). MP: 90.2–91.3° C. IR (cm⁻¹): 3280(br), 1702 (s), 1562 (m), 1210 (s), 1180 (s). ¹H NMR (400 MHz, DMSO-d₆):δ 3.22 (s, 3), 3.37–3.42 (m, 6), 3.49–3.57 (m, 10), 3.72 (t, 2, J=4.6),4.07 (t, 2, J=4.6), 5.52 (s, 1), 6.91 (d, 2, J=8.7), 7.29 (d, 2, J=8.7),9.51 (t, 2, J=5.5). ¹³C NMR (DMSO-d₆): δ 39.22, 58.00, 62.57, 67.12,68.91, 69.59, 69.78, 69.93, 71.26, 100.57, 113.93, 116.14 (q, J=241),127.72, 130.26, 156.40 (q, J=36), 158.52. Calcd: [M]⁺ (C₂₂H₃₀F₆N₂O₈)m/z=564.1906. Found FAB-HRMS: [M]⁺ m/z=564.1922. Anal. Calcd. forC₂₂H₃₀F₆N₂O₈: C, 46.81; H, 5.36; N, 4.96. Found: C, 46.97; H, 5.38; N,4.72.

N,N′-Bisacryloyl-di-(2-aminoethoxy)-[4-(1,4,7,10-tetraoxaundecyl)phenyl]methane(606). Compound 605 (4.0 g, 7.1 mmol, 1 equiv) and 6 M NaOH (30 mL) wereadded to dioxane (20 mL) and the reaction mixture was stirred at roomtemperature for 7 h. Complete removal of the acetamide groups wasdetermined by TLC using ninhydrin staining. Upon completion, thereaction mixture was cooled to 0° C. and triethylamine (3 mL) was added.Acryloyl chloride (12 mL, 0.15 mol, 21 equiv) and triethylamine (36 mL,0.26 mol, 36 equiv) were added in small alternating portions whileperiodically monitoring the pH to maintain it above 7. A 10% K₂CO₃ inwater solution (30 mL) was added, and the reaction mixture was stirredfor 10 minutes before extracting the product with four 200 mL portionsof ethyl acetate. The organic layers were combined, dried, andevaporated and the product crystallized from ethyl acetate/hexane,yielding 2.05 g (58%) of the crosslinker as a white solid. MP:83.6–85.5° C. IR (cm⁻¹): 3293 (br), 1665 (s), 1562 (s), 1245 (s), 1101(s). ¹H NMR (400 MHz, CDCl₃): δ 3.38 (s, 3), 3.53–3.76 (m, 16), 3.86 (t,2, J=4.8), 4.14 (t, 2, J=4.8), 5.44 (s, 1), 5.65 (dd, 2J=17, J=2), 6.15(dd, 2, J=17, J=10), 6.22 (s, 2), 6.30 (dd, 2, J=10, J=2), 6.91 (d,2J=8.8), 7.32 (d, 2, J=8.8). ¹³C NMR (DMSO-d₆): δ 38.71, 58.04, 63.80,67.10, 68.93, 69.61, 69.80, 69.94, 71.27, 100.83, 113.91, 125.10,127.86, 130.62, 131.69, 158.45, 164.72. Calcd: [M+H]⁺ (C₂₄H₃₇N₂O₈)m/z=481.2549. Found FAB-HRMS: [M+H]⁺ m/z=481.2544. Anal. Calcd. forC₂₄H₃₆N₂O₈: C, 59.99; H, 7.55; N, 5.83. Found: C, 59.86; H, 7.75; N,5.77.

EXAMPLE 5 Synthesis of Bisacrylamide Tetraglyme Acetal Crosslinker

FIG. 7 shows the synthetic pathway of another crosslinker, in whichR¹=(a) of FIG. 2 and R²=aryl-O-[CH₂—CH₂—O]₄—CH₃, referred to as“tetraglyme.” This crosslinker has properties, as compared to the othercrosslinkers disclosed herein, including, but not limited to, increasedprotein loading and loading efficiency, better dispersability insolution, and higher T-cell activation achieved.

1-Chloro-3,6,9,12-tetraoxatridecane (707). This compound was preparedaccording to the procedure reported by Schafheute et al. ¹⁶ IR (cm⁻¹):2875 (s), 1149 (s). ¹H NMR (300 MHz, CDCl₃): δ 3.38 (s, 3), 3.55 (t, 2,J=4.6), 3.75 (t, 2, J=5.9), 3.60–3.67 (m, 12). ¹³C NMR (CDCl₃): δ 42.36,58.60, 70.11, 70.19, 70.21, 70.24, 70.96, 71.55. Calcd: [M+H]⁺(C₉H₂₀O₄Cl) m/z=227.1050. Found FABHR-MS: [M+H]⁺ m/z=227.1045. Anal.Calcd. for C₉H₁₉O₄: C, 47.68; H, 8.45. Found: C, 47.83; H, 8.62.

p-(1,4,7,10,13-Pentaoxatetradecyl)benzaldehyde (708). Chloride 707 (4.85g, 21.45 mmol, 2 equiv.) and p-hydroxybenzaldehyde (1.31 g, 10.72 mmol,1 equiv) were dissolved in dry THF (5 mL). K₂CO₃ (1.49 g, 10.75 mmol, 1equiv.) was added followed by 18-crown-6 (50 mg, 0.19 mmol, 0.01 equiv.)and KI (50 mg, 0.301 mmol, 0.01 equiv.). The reaction mixture wasstirred and heated at reflux for 24 hours. The reaction mixture wascooled down to room temperature and water (50 mL) was added. The productwas extracted with ethyl acetate (3×50 mL) and the combined organiclayers were dried and evaporated. The oil was loaded onto a silica gelcolumn and eluted with a 1:1 ethyl acetate/hexane mixture to afford 2.41g (72%) of 704 as a clear oil. IR (cm⁻¹): 1693 (s), 11.32 (s). ¹H NMR(300 MHz, CDCl₃): δ 3.33 (s, 3), 3.50 (t, 2, J=4.5), 3.72–3.58 (m, 10),3.86 (t, 2, J=4.8), 4.18 (t, 2, J=4.8), 6.99 (d, 2, J=8.7), 7.79 (d, 2,J=8.6), 9.80 (s, 1). ¹³C NMR (CDCl₃): δ 58.82, 67.59, 69.26, 70.32,70.41, 70.43, 71.73, 114.70, 129.84, 131.74, 163.68, 190.59. Calcd:[M+H]⁺ (C₁₆H₂₅O₆) m/z=313.1651. Found FABHR-MS: [M+H]⁺ m/z=313.1643.

p-(1,4,7,10,13-Pentaoxatetradecyl)benzylacetal-bistrifluoroacetamide(709). Aldehyde 708 (0.88 g, 2.8 mmol, 1 equiv.) andN-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (4.02 g, 21.3 mmol, 7.6equiv.) were dissolved in dry THF (6 mL). p-Toluenesulfonic acid (95 mg,0.5 mmol, 0.16 equiv.) and 5 Å molecular sieves (7 g) were added. Thereaction mixture was stirred overnight and was quenched withtriethylamine (0.5 mL, 3.6 mmol, 1.3 equiv.). The reaction mixture wasfiltered. Water (50 mL) was added. The product was extracted into ethylacetate (5×50 mL) and the solvent was evaporated. In order to remove theexcess alcohol, benzoyl chloride (1.82 mL, 31.4 mmol, 1 equiv.),triethylamine (4.37 mL, 62.8 mmol, 2 equiv.), and dry THF (10 mL) wereadded. The reaction mixture was stirred at room temperature for 1 hour.Water (100 mL) was added and the product was extracted into ethylacetate (5×100 mL) and the solvent was evaporated. The remaining oil wasloaded onto a silica gel column and eluted with a 3:7 ethylacetate/hexane mixture followed by a 4:1 ethyl acetate/hexane mixture toafford 1.11 g (64%) of 709 as a white solid. MP: 74.6–75.0° C. IR(cm⁻¹): 3292 (br), 1701 (s), 1560 (m), 1209 (s), 1178 (s). ¹H NMR (400MHz, DMSO-d₆): δ 3.21 (s, 3), 3.34–3.41 (m, 6), 3.46–3.58 (m, 14), 3.72(t, 2, J=4.6), 4.07(t, 2, J=4.6), 5.52 (s, 1), 6.91 (d, 2, J=8.4), 7.29(d, 2, J=8.4), 9.53 (t, 2, J=5.6). ¹³C NMR (DMSO-d₆): δ 39.22, 58.01,62.56, 67.12, 68.89, 69.56, 69.76, 69.81, 69.91, 71.26, 100.54, 113.92,115.19 (q, J=288), 127.72, 130.25, 156.38 (q, J=36), 158.51. Calcd:[M+H]⁺ (C₂₄H₃₄F₆N₂O₉) m/z=608.2168. Found FABHR-MS: [M]⁺m/z=608.2153.Anal. Calcd. for C₂₄H₃₄F₆N₂O₉: C, 47.37; H, 5.63; N, 4.60. Found: C,47.20; H, 5.84; N, 4.54.

p-(1,4,7,10,13-Pentaoxatetradecyl)benzylacetal-bisacrylamide (710).Compound 709 (400 mg, 0.66 mmol, 1 equiv.) and 6M NaOH (2.8 mL) wereadded to dioxane (1.8 mL) and the reaction mixture was stirred at roomtemperature for 3.5 hours. Complete removal of the acetamide groups wasmonitored by TLC and ninhydrin staining. Upon completion, the reactionmixture was cooled down to 0° C. and triethylamine (0.6 mL) was added.Acryloyl chloride (1.1 mL, 13.8 mmol, 21 equiv.) and triethylamine (5.6mL, 40.4 mmol, 61 equiv.) were added in small alternating amounts whileperiodically monitoring the pH so that it did not go below 7. A 10%K₂CO₃ in water solution (40 mL) was added and the reaction was stirredfor 10 minutes before extracting the product into ethyl acetate (6×40mL). The organic layers were dried and evaporated to afford a yellowoil. The oil was loaded onto a silica gel column and eluted with a 2:1ethyl acetate/hexane mixture followed by ethyl acetate and a 1:9methanol/ethyl acetate mixture to afford 200 mg (58%) of 710 as a whitesolid. MP: 62.0–63.0° C. IR (cm⁻¹): 3302 (br), 1657 (s), 1541 (m), 1102(s). ¹H NMR (300 MHz, DMSO-d₆): δ 3.21 (s, 3), 3.29 (t, 2, J=4.5),3.39–3.55 (m, 18), 3.72 (t, 2, J=4.6), 4.07 (t, 2, J=4.6), 5.49 (s, 1),5.56 (dd, 2, J=10, J=2), 6.10 (dd, 2, J=17, J=2), 6.30 (dd, 2, J=17,J=10), 6.90 (d, 2, J=8.6), 7.32 (d, 2, J=8.6), 8.20 (t, 2, J=5.5). ¹³CNMR (DMSO-d₆): δ 38.67, 58.01, 63.77, 67.07, 68.88, 69.54, 69.74, 69.78,69.89, 71.24, 100.80, 113.88, 125.06, 127.83, 130.60, 131.67, 131.87,158.42, 164.68. Calcd: [M+Li]⁺ (C₂₆H₄₀N₂O₉Li) m/z=531.2893. FoundFABHR-MS: [M+Li]⁺ m/z=5.31.2883. Anal. Calcd. for C₂₆H₄₀N₂O₉: C, 59.53;H, 7.69; N, 5.34. Found: C, 59.19; H, 7.63; N, 5.04.

EXAMPLE 6 Hydrolysis Kinetics of a Bisacryloyl Acetal Crosslinker

A key aspect of the bisacryloyl acetal crosslinker is its hydrolysiskinetics. The crosslinker is designed to be stable at the physiologicalpH of 7.4 but it undergoes rapid hydrolysis at acidic pHs. This isdemonstrated by measurements performed with the bisacrylamidemethoxybenzaldehyde acetal crosslinker 110 at pH 5.0 and at pH 7.4. AtpH 5.0, the crosslinker hydrolyzes rapidly, with a half-life of 5.5minutes, whereas at pH 7.4 the half-life is 24 hours.

A stock solution of the bisacrylamide methoxybenzaldehyde acetalcrosslinker (10 mg/mL) in THF was prepared and 10.5 μL (1×10⁻⁴ mol/L)was added to a 3.0 ml PBS solution at either pH 5.0 or 7.4, in aspectrophotometer cuvette. The hydrolysis of the acetal was monitored bymeasuring the absorbance of the 4-methoxybenzaldehyde, produced by theacetal hydrolysis, at 280 nm. The percentage of hydrolysis wascalculated by the following equation: percent hydrolysis (%) at timei=(A_(i)−A₀)/(A_(∞)−A₀)×100%, where A=Absorbance at 280 nm. At pH 5.0,95% hydrolysis was complete in 20 minutes, with about 50% hydrolysis inless than 10 minutes.

The acceleration of the hydrolysis kinetics of this crosslinker from pH7.4 to pH 5.0 was apparent. The hydrolysis of such benzaldehyde acetalsis proportional to the hydronium ion concentration, which increases 250fold between pH 7.4 and pH 5.0. The second order hydrolysis rateconstant of a bisacrylamide methoxybenzaldehyde acetal crosslinker is5,610 min⁻¹mole⁻¹. The hydrolysis rate constant of this crosslinker is 5times slower than the hydrolysis rate constant of the dimethyl acetal ofmethoxy benzaldehyde. This rate reduction of the bisacrylamidemethoxybenzaldehyde acetal crosslinker may be due to the electronwithdrawing effects of the amide groups on the alkoxy portion of theacetal and is beneficial because it increases shelf life.

EXAMPLE 7 Synthesis of Acid Degradable Microgel Particles EncapsulatingBioactive Material Using Inverse Microemulsion Technique

Microgel particles were synthesized by inverse microemulsionpolymerization, according to the procedure described by Kriwet, B.;Walter, E.; Kissel, T.; J. Control Release, 1998, (56), 149–158. A keyissue in the synthesis of microgels by inverse emulsion polymerizationis the aqueous solubility of the monomers. Several different emulsionpolymerization procedures were attempted with the bisacrylamide acetalcrosslinker, using different organic phases and surfactant blends.Inverse polymerizations with toluene/chloroform as the continous phaseand pluronic F-68 as the surfactant were unsuccessful. However, inversepolymerization with hexane as the continous phase and of SPAN™ 80(sorbitan monooleate), TWEEN™ 80 (polyethyleneglycol-sorbitanmonooleate, AOT and Brij as surfactants were successful in producingmicrogels.

The improved performance of the bisacrylamide methoxy benzaldehydeacetal crosslinker 110 in hexane water versus chloroform-toluene/wateris potentially explained by the lower solubility of the crosslinker inhexane versus chloroform-toluene. For example, the bisacrylamide methoxybenzaldehyde acetal crosslinker 110 has water/hexane partition ratio of10,000:1, in contrast the water/toluene-chloroform partition ratio isonly 1:1, suggesting that in the water/chloroform-toluene polymerizationa large fraction of the crosslinker is lost in the organic phase.

The following protocol illustrates the preparation of the presentmicrogel particles encapsulating a bioactive material (albumin). Theseparticles are discussed further in connection with Example 11. Table 1of Example 11 sets forth the components of three different microgelparticles. In this example, microgel particles with crosslinking ratiosranging from 1.6%–12.8% were prepared using this inverse emulsionpolymerization procedure with bisacrylamide triglyme acetal crosslinker606 of Example 4 and acrylamide.

The organic phase of the polymerization consisted of 5 mL of hexanecontaining 150 mg of a 3:1 weight ratio of SPAN™ 80 and TWEEN™ 80. Theaqueous phase of the polymerization consisted of 125, 200, or 225 mg ofacrylamide and 25, 50 or 125 mg of the bisacrylamide triglyme acetalcrosslinker of Example 4 (with a combined weight of 250 mg), dissolvedin 0.5 ml of sodium phosphate buffer pH 8.0 300 mM sodium phosphate, 12mg of the free radical initiator potassium peroxodisulfate and 5.4 or5.7 mg ovalbumin. The aqueous and organic phases were deoxygenated withnitrogen. An inverse emulsion between the organic and aqueous phases wasformed by mixing them and then sonicating for 30 seconds. Polymerizationof the inverse emulsion was then initiated while stirring with amagnetic bar by adding 10 μl of N,N,N′,N′-tetramethylethylene diamine.The stirred polymerization was allowed to proceed for 10 minutes at roomtemperature.

After polymerization, the mixture was centrifuged at 2800 rpm for 10minutes and the solvent was decanted off. The microgels were carefullywashed with hexane (2×20 mL), acetone (4×20 mL) and isolated bycentrifugation at 2,800 rpm for 10 minutes. The recovered microgels werevacuum dried overnight and analyzed by scanning electron microscopy (WDXISI-ds130C, Microspec Corp.) at 15 kV. A scanning electron microscopy(SEM) image of the particles (not shown) showed that the particle sizevaried between 200 nm and 500 nm in the dry state. This sizedistribution is suitable for protein delivery to APCs, which internalizeparticles between 200 nm–5 μm by phagocytosis.

EXAMPLE 8 Synthesis of Bisacrylamide Nitrophenylcarbonate AcetalCrosslinker

The goal of synthesizing microgel particles with dextran was to generatemicrogel particles that would degrade to a low molecular weightexcretable backbone. Referring now to FIG. 8, a bisacrylamidenitrophenylcarbonate acetal crosslinker 836 can be synthesized by thesynthesis steps as shown in FIG. 8A.

The synthesis of the bisacrylamide nitrophenylcarbonate acetalcrosslinker 836 was accomplished in four steps. The first step wasalkylation of hydroxy benzaldehyde 810 with bromo-ethylacetate 820,using potassium carbonate and 18-6 crown as the base. The product waspurified by a small silica gel column, and this synthesis could beperformed on a 20 gram scale. The second step was acetal formationbetween hydroxy-ethyl trifluoroacetamide 830 and the benzaldehydeacetate 830 from previous, using p-toluene sulfonic acid as a catalyst.The product, bistrifluoroacetamide methoxyphenyl-ethyl acetate 834 (50%yield), was purified by crystallization from ethyl acetate and hexane,using 5 A molecular sieves and could be synthesized on a multigramscale. The bistrifluoroacetamide methoxyphenyl-ethyl acetate 834 wasthen deprotected in 6N NaOH and reacted with acryloyl chloride, thereaction product was purified by crystallization from ethyl acetate, togive the hydroxyl compound 835. This reaction needs to be performed in6N NaOH otherwise the hydroxyl will react with the acryloyl chloride.The compound 835 was converted to the bisacrylamide nitrophenylcarbonateacetal crosslinker 836 by reacting with para-nitrochloroformate, in thepresence of triethyl amine. The crosslinker 836 was purified bycrystallization from ethyl acetate.

EXAMPLE 9 Synthesis of Dextran Microgels

Dextran microgel particles were made using the bisacrylamidenitrochloroformate acetal crosslinker 836 of Example 8 using inversemicroemulsion polymerization as described in Example 7. Referring now toFIG. 8B, the bisacrylamide nitrochloroformate acetal crosslinker 836 wasmodified to the dextran acetal crosslinker 840 by introducing an aminehandle on dextran and then reacting this activated dextran 838 with thecrosslinker 836. The amine was introduced onto the dextran by activatingthe dextran hydroxyls with para-nitrochlorofomate and then reacting itwith diamino-diethylene glycol. ¹H-NMR indicated that 1 out every sixhydroxyls were functionalized with the amine handle. The purification ofthe dextran products was performed by precipitating the reaction inethanol. The final dextran acetal crosslinker 840 was synthesized byreacting the amine functionalized dextran 838 with the crosslinker 836,the product 840 was purified by precipitation in ethanol and sizeexclusion chromatography. H-NMR and U.V. spectroscopy indicated thatapproximately 1 out 6 of the sugars reacted with the crosslinker 836.

Referring now to FIG. 8C, dextran microgels were made under inversemicroemulsion polymerization conditions using SPAN 80/TWEEN 80 assurfactants and hexane as the oil phase. Upon addition of S₂O₈K₂(potassium persulfate), 300 mg of the bisacrylamide-dextran-acetalcrosslinker 840, in the presence of 1 mg DNA, copolymerized to entrapthe DNA and formed loaded dextran microgels which are biodegradable asshown in FIG. 10. An SEM image of the microgels showed that the shape ofthe dextran microgels are not spherical, but nevertheless are individualmicroparticles. The number of dextran molecules in this example (X inFIG. 8B) should preferably be between 3 to 555 sugar molecules, with aMW of no more than 100,000.

EXAMPLE 10 Synthesis of Bisacrylamide-Dextran-Acetal Crosslinker

A bisacrylamide-dextran-acetal crosslinker 920 can be synthesized by thesynthesis steps as shown in FIGS. 9A–9B. Referring now to FIG. 9A,hydroxy benzaldehyde 902 is reacted with 1,3 bromo-propyl-chloride inthe presence of K₂ CO₃, 18-6 Crown Ether, and THF, resulting inbenzaldehyde-4-methoxy-propyl chloride 844 at 30% yield. The azide isformed by reacting compound 904 with NaN₃, DMF at 90–100° C., yieldingazidopropyl benzaldehyde 906 in 70% yield. The acetal linkage is made byreacting the second intermediate, azidopropyl benzaldehyde 906, with (2)molecules of hydroxy-ethyl trifluoroacetamide, in the presence ofp-toluenesulfonic acid, THF, using 5 A molecular sieves to yield thebistrifluoroacetamide methoxyphenyl-propyl-azide-acetal 908 at 49%yield. This intermediate azide-acetal 908 is then reduced to an amineacetal by PPh₃, THF, TEA, H₂O to yield a bistrifluoroacetamide amineacetal 910.

Referring now to FIG. 9B, the bistrifluoroacetamide amine acetal 910 isthen reacted with para-nitrochloroformate-activated dextran 914 in DMSOto yield a dextran-trifluoroacetamide acetal. The activated dextran 914is prepared as described in Example 9, wherein an amine handle ondextran was introduced to produce activated dextran 838. The amine wasintroduced onto the dextran by activating the dextran hydroxyls withpara-nitrochlorofomate and then reacting it with diamino-diethyleneglycol. ¹H-NMR indicated that 1 out every six hydroxyls werefunctionalized with the amine handle. The purification of the dextranproducts was performed by precipitating the reaction in ethanol. Theproduct is then purified by ether/ethanol precipitation and gelpermeation chromatography to yield abistrifluoriacetamide-dextran-acetal 916.

Referring now to FIG. 9C, after purification, the bisacrylamide dextranacetal 920 was synthesized in two steps by first reacting thebistrifluoriacetamide-dextran-acetal 916 with K₂ CO₃, MeOH and H₂O toyield a bisamine-dextran-acetal 918. The addition of pyridine and pH 10buffer in the presence of acryloyl chloride to maintain pH then yields abisacrylamide-dextran-acetal crosslinker 920.

Refering now to FIG. 9D, dextran microgels can be made using the inversemicroemulsion polymerization conditions of Example 7. Upon addition of aradical source like potassium persulfate to the bisacrylamide dextranacetal crosslinker 920 in the presence of bioactive materials, thecrosslinker will polymerize and trap the bioactive material 40 and formloaded dextran microgels which are biodegradable as shown in FIG. 10.

The number of dextran molecules attached should preferably be between 3to 555 sugar molecules, with a MW of no more than 100,000.

EXAMPLE 11 Increased Protein Loading

The bisacryloyl acetal crosslinker used to synthesize acid degradableprotein loaded microgels influences the loading efficiency of themicrogels. The bioactive material is physically entrapped in themicroparticle by polymerizing the polymerizable groups and crosslinkerin the presence of the bioactive material. A key parameter in thesynthesis of protein-loaded microgels is their “pore size”, which needsto be smaller than the radius of the protein or other bioactive materialfor efficient encapsulation.

Ovalbumin was chosen as the model protein for the encapsulation studiesbecause numerous immunological assays have been developed for thisprotein. Ovalbumin labeled with Cascade Blue was encapsulated inmicrogels containing 1.6, 3.5 and 12.8 mole percent of bisacrylamidetriglyme-acetal crosslinker 606. The results of the proteinencapsulation experiment are listed in Table 1, with protein loadingsvarying from 9–11 μg of protein per mg of microgel. Based on thestarting protein/monomers ratio, this represents about 50% encapsulationefficiency.

Protein Encapsulation Measurements. 2 mg of each microgel particlesample (see Table 1), containing Cascade Blue labeled Ovalbumin, wasdispersed in 0.5 mL of pH 8.0 300 mM sodium phosphate buffered water bysonication for 5 minutes. The microgel samples were centrifuged for 5minutes and the supernatant was pipetted off to remove any unboundprotein. The washed microgels were then hydrolyzed in 300 mM sodiumacetate buffered water (pH 1.6, 500 μL). After complete hydrolysis ofthe microgel particles, the quantity of encapsulated protein wasdetermined by fluorescence spectroscopy, excitation at 405 nm, emissionat 460 nm. The protein concentration of each microgel sample wascalculated by fitting the emission to a calibration curve made fromknown concentrations of Cascade Blue labeled Ovalbumin. The loadingefficiency measurements may be lower than actual amount because theremay protein entrapped in microgels that do not centrifuge down and thuscannot be recovered.

The encapsulation efficiency obtained with the bisacrylamidetriglyme-acetal crosslinker was similar to that observed by others forthe encapsulation of ovalbumin in non-degradable microgels composed of2.5 mole percent methylene bisacrylamide and 97.5 mole percentacrylamide (O'Hagan, D. T.; Palin, K.; Davis, S. S.; Artursson, P.;Sjoholm.; Vaccine. 1989, (7), 421–424. ). The encapsulation efficiencyof the microgel particles made with the bisacrylamide triglyme-acetalcrosslinker did not change appreciably with the crosslinking ratio asshown by the amounts of encapsulated albumin per mg of microgel particle(μg/mg) in Table 1. This lack of correlation between crosslinking ratioand protein encapsulation has been previously observed by Ekman et al.for the encapsulation of human serum albumin in non-degradable microgelscomposed of methylene bisacrylamide and acrylamide (Ekman, B. et al.,(1976) Biochemistry 15, 5115–5120).

TABLE 1 Cascade Wt. % Mole Blue Encapsulated Yield Percent Cross- Acryl-Labeled Albumin/ of Sam- Cross- linker amide Albumin Particle Wt. microple linking (mg) (mg) (mg) (μg/mg) gels A 1.6 25 225 5.4 10.4 38.0 B 3.550 200 5.7 11.1 61.0 C 12.8 125 125 5.4 9.5 43.0

The protein loading efficiency of the gel particles polymerized with1.6% crosslinking with the bisacrylamide triglyme-acetal crosslinker 606of Example 4 was compared to the protein loading efficiency of themicrogel particles polymerized with the 1.6% of the bisacrylamidetetraglyme-acetal crosslinker 710 of Example 5. The crosslinkers werecopolymerized with acrylamide in a PBS buffer containing thefluorescently labeled protein FITC-Albumin (1 mg/ml). Table 2 lists theresulting amount of ovalbumin encapsulated per milligram of microgelparticles.

TABLE 2 Cascade Blue- Triglyme Encapsulated Tetraglyme EncapsulatedAlbumin in Albumin/Microgels Albumin/Microgels Polymerization (mg)(μg/mg) (μg/mg) 21.0  9.5 44.3  67.2 22.0 — 133.0 62.6 135.6 183.9 —154.8

EXAMPLE 12 pH Dependant Release of Encapsulated Bioactive Material byHydrolysis

The bisacrylamide triglyme acetal crosslinker 606 was used to synthesizeacid degradable protein loaded microgels according to the conditionsdescribed in Example 7. 2 mg of each microgel sample from Table 1containing Cascade Blue labeled Ovalbumin was dispersed in 0.5 mL of pH8.0 300 mM sodium phosphate buffered water by sonication for 5 minutes.The microgel samples were centrifuged for 5 minutes and the supernatantwas pipetted off to remove any unbound protein. The recovered pellet wasthen redispersed into either 300 mM acetic acid buffered water (pH 5.0,500 μL) or 300 mM sodium phosphate buffer (pH 7.4, 500 μL). Thesolutions were incubated at 37° C. in a heating block for each timepoint. The percentage of protein released at a given time point wasdetermined by centrifuging the microgel sample for 5 minutes, isolatingthe supernatant from the pellet and comparing the fluorescence of thesupernatant (released protein) with that of the pellet (protein still inmicrogels), excitation at 405 nm, emission at 460 nm. The recoveredpellet was hydrolyzed in pH 1.6 300 mM acetic acid, before measuring itsfluorescence. The background emission of each buffer was measured andsubtracted from all of the readings.

The release of protein from the microgels was measured at pH 5.0 and 7.4to understand their behavior in the environments of the phagosome andblood, respectively (data not shown). Microgels made with bisacrylamidetriglyme acetal crosslinker 606 release their contents much faster at pH5.0 than at pH 7.4. For example, at pH 5.0, after 5 hours, the 1.6%crosslinked microgels released 80% of encapsulated ovalbumin, whereas atpH 7.4, only 10% was released. This pH dependency is caused by the acidsensitivity of the crosslinker, which hydrolyzes rapidly at pH 5.0, thusincreasing the effective “pore size” of the microgels and the diffusionrate of proteins out of the microgels. In contrast, at pH 7.4, thebisacrylamide triglyme acetal crosslinker remains intact and themajority of encapsulated proteins are retained. A small percentage ofencapsulated proteins are initially released at pH 7.4. This is likelydue to the vigorous vortexing and sonication procedures used to dispersethe microgel particles, which could dislodge proteins loosely entrappedwithin the particles.

EXAMPLE 13 Class I Antigen Presentation Assays

The LacZ MHC Class I antigen presentation assay, as described bySanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369–376, wasperformed with the microgel particles made with the bisacrylamidetetraglyme acetal crosslinker 710 to test their ability to deliverproteins into APCs for Class I antigen presentation. This experimentuses the LacZ B3Z hybridoma, which is a CTL that recognizes the peptidesequence, SIINFEKL, from ovalbumin, complexed with the MHC Class Imolecule H-2K^(b). This hybridoma produces β-galactosidase afterencountering APCs that present SIINFEKL as a Class I antigen, thusallowing Class I antigen presentation to be quantified by measuringβ-galactosidase activity.

A proper control would be to compare the amount presented by cells whenincubated with the SIINFEKL peptide which is directly displayed on theantigen presenting cells and not delivered to the cytoplasm of the cellsfirst. A maximum absorbance of 0.25 is observed with the SIINFEKLpeptide, which results in 100% T-cell activation. At about 0.4 mg ofparticle/mL, the particles made with a 1:1 ratio of acrylamide to thebisacrylamide tetraglyme crosslinker of Example 5, shows an absorbanceclose to that of 0.25 at which 100% T-cell activation occurs.

The results of the Class I antigen presentation assay show that greaterT-cell activation is seen for albumin loaded particles vs. free protein.APCs incubated with free ovalbumin are not able to activate CTLs,indicating that these APCs are unable to present free ovalbumin as aClass I antigen. This is presumably because ovalbumin endocytosed by theAPCs, is sequestered in lysosomes, and does not have access to the APCcytoplasm. In contrast, APCs incubated with ovalbumin encapsulated inthe microgel particles, can efficiently activate CTLs. Ovalbuminencapsulated in the microgels is several orders of magnitude moreefficient than free ovalbumin at inducing the activation of CTLs, forexample, 1 μg/ml of ovalbumin encapsulated in the microgels gives T cellactivation levels that are 3 times greater than 1 mg/ml of freeovalbumin (the U.V. absorbance resulting from activation with 1 mg/ml offree ovalbumin was only 0.037 versus 0.1106 for activation with 1 μg/mLof ovalbumin encapsulated in the microgels). Thus the acid degradablemicrogels are capable of delivering protein antigens into APCs for ClassI antigen presentation.

Higher protein loading was shown to lead to an increase in antigenpresentation. For example, the absorbance taken for 0.1 mg particle/mLof particles, made with the biscarylamide triglyme acetal crosslinker ofExample 4, having a protein loading capacity of 22 μg protein/mg ofparticle, is close to the same absorbance for 0.5 mg particle/mL ofparticles, made with the same but greater percentage of crosslinker, andhaving a 9.5 μg/mg loading capacity. There may be a point where there isa maximum level of antigen presentation as shown by the same absorbanceof about 0.33 with 0.5 mg/mL of particles having 22.0 and 62.6 μgprotein/mg of particle loading capacity. In contrast the biscarylamidetetraglyme acetal crosslinker of Example 5 has a 135.6 μg/mg loadingcapacity and an absorbance of 0.38, which is almost twice the absorbanceof the SINFEKL peptide in this antigen presentation assay.

EXAMPLE 14 Toxicity of Microgels Made with Bisacrylamide AcetalCrosslinker

The toxicity of bioactive material loaded microgels was measured withthe yellow tetrazolium salt,3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), assayusing RAW 309.CR1 macrophage cells (ATCC No. TIB-69, American TypeCulture Collection, Manassas, Va.). The cells are incubated withmicrogel particles in DMEM media with 10% F.B.S. The microgels wereaspirated from the cells, and they were then washed several times withPBS and allowed to grow for 24–48 hours. The cell viability isdetermined by measuring the absorbance of the reduced MTT reagent usingthe protocol described in Freshney et al. (Freshney, I. R. (1994)Culture of animal cells, Wiley-Liss, Inc, New York, N.Y.) as compared toa control. MTT (yellow) is reduced metabolically by healthy cells inpart by the action of dehydrogenase enzymes in mitochondria, to generatepurple formazan crystals, which are solubilized by the addition of adetergent and the absorbance is measured at 570 nm. Thus, themeasurement of the ability of cells to reduce the MTT reagentmetabolically is a measurement of the health of the cell population.

RAW 309.CR1 macrophage cells were split at 5×10⁴ cells per well in a 96well plate and allowed to grow overnight. The cells were then incubatedwith the microgel particles (1.6% crosslinked, sample A from Table 1 inExample 11) with variable amounts of loaded ovalbumin for 24 hours inDMEM media with 10% F.B.S. The microgels were aspirated from the cells,and they were then washed several times with PBS and allowed to grow foranother 24 hours.

The cell viability was determined by measuring the absorbance of thereduced MTT reagent. The MTT assay was performed using 0.5, 1, 2.5 and 5mg particles/mL serum in each well with a microgel loading of 10micrograms protein/mg microgel particle. After 24 hours, there werealmost 100% viable cells remaining in the 0.5 mg particles/mL, 90%viable cells remaining in the 1 mg protein/mg particles, about 80%viable cells remaining in the 2.5 mg particles/mL, and about 80% viablecells remaining in the 5 mg particles/mL. Thus, it can be found that themicrogels of the invention are not toxic to mammalian cells because morethan 50% of the cells remain viable.

The effect of variable amounts of crosslinking on viability of cells wasalso observed. RAW 309.CR1 macrophage cells were split at 5×10⁴ cellsper well in a 96 well plate and allowed to grow overnight. The cellswere then incubated with the microgel particles made with thebisacrylamide tetraglyme acetal crosslinker (1.6% crosslinked, 9:1ratio, sample A and 12.8% crosslinked, 1:1 ratio sample C from Table 1in Example 11) with variable amounts of loaded oavalbumin for 20 hoursin DMEM media with 10% F.B.S. The particles were aspirated from thecells, and they were then washed several times with PBS and allowed togrow for another 24 hours.

The MTT assay was performed using 0.5, 1, 2.5 and 5 mg protein/mL serumin each well with a particle loading of 10 micrograms protein/mgmicroparticle After 24 hours, cells incubated with the particles having12.8% crosslinker showed 80% viable cells remaining after exposure to0.1 mg/mL of particles, 74% viable cells remaining in 0.5 mg/mLparticles, 62% viable cells remaining in 1.0 mg/mL particles, 63% viablecells remaining in 2.5 mg/mL particles, and 47% viable cells remainingin 5 mg/mL particles. After 24 hours, cells incubated with the particleshaving 12.8% crosslinker showed 70% viable cells remaining in 0.5 mgparticles/mL, 63% viable cells remaining in 0.5 mg particles/mL, 62%viable cells remaining in the 1 mg protein/mg particles, about 62%viable cells remaining in the 1 and 2.5 mg protein/mL particles, andabout 52% viable cells remaining in the 5 mg protein/mL particles.

EXAMPLE 15 Cytoplasmic Release of Bioactive Material from Microgels

Microgel particles were made with acrylamide and bisacrylamide triglymeacetal crosslinker 606 encapsulating fluorescently labeled dextran(because it easier to label and observe than fluorescence-labeled DNA)and fed to macrophage cells. When a bisacrylamide methylenenondegradable crosslinker is used, the fluorescence is more localizedshowing that when nondegradable microgels have been taken up by thecells, they remain sequestered in the lysosome without a mechanism ofrelease. When the acid degradable bisacrylamide triglyme acetalcrosslinker is used to make the microgels, the fluorescence is morediffuse within the cytoplasm of cells, which is indicative ofcytoplasmic release of the microgel contents.

EXAMPLE 16 Excretion of Degraded Particles

Polymers with high MW are not easily excreted from body, thereforeanother aspect of the invention is to make microgel particles thateasily and safely excreted by the body after being degraded in theacidic cellular compartment. Light scattering results in Table 3 showedthe following for microgel particles made without protein afterhydrolysis and purification by dialysis with a minimum size of 13,000 MWas a cutoff size:

TABLE 3 Crosslinking Mole Ratio dn/dc Mw 9:1 acrylamide/triglymecrosslinker 0.177 498,000 1:1 acrylamide/triglyme crosslinker 0.169604,000

Referring now to FIG. 10, in a preferred embodiment, dextran microgelparticles were made according to Example 9 using the crosslinker ofExample 8. After degradation of the microgels, (activated) dextran of10,000 MW is easily secreted from the body and should exhibit notoxicity problems because it is a sugar. The bioactive material 40 isreleased along with the linker group upon hydrolysis.

However, one aspect of using dextran microgels that was observed is thatprotein loading must be about 10 μg protein/mg particle before theantigen presentation assay of Example shows absorbance of 0.25 which isthe target absorbance at which there is 100% T-cell activation. Dextranmicrogel particles also enhance antigen presentation versus free proteinbut about ⅓ as efficient as using acrylamide as the polymerizing group.One other concern with dextran microgels is the dispersability of theparticles in solution because dextran may not be sufficientlyhydrophilic.

EXAMPLE 17 Gel Particles Encapsulating Plasmid DNA

Synthesis. Synthesis of gel particles encapsulating plasmid DNA was asfollows. Plasmid DNA (pSV-β-gal vector, 6820 bp, Amp resistant) is addeddirectly to the aqueous phase of an inverse microemulsion. The procedureis directly analogous to that for the protein loaded microgel particlesmade in Example 7. The organic phase consisted of hexane with 3% of thesurfactants: 3/1 SPAN 80/TWEEN 80. Acrylamide monomer and thebisacrylamide triglyme acetal crosslinker (in a 4/1 mass ratio),potassium persulfate, and 250 ng plasmid DNA were dissolved in 300 mMPBS, pH 8.0. The two phases are combined and sonicated for 30 sec, atwhich point, TMEDA (tetramethylethylenediamine) was added, and thepolymerization allowed to proceed for 10 min. The microgels werecollected by centrifugation (10 min×3000 RPM) and washed once withhexane and twice with acetone, then dried under vacuum overnight.

Loading Efficiency. To examine the loading, the microgels were suspendedin pH 7.4 buffer (300 mM PBS) to a concentration of 5 mg/mL. They werethen collected by centrifugation, and the supernatant was removed bypipet. This step serves to remove any DNA that is adsorbed to thesurface of the microgels but is not actually incorporated inside. Themicrogels were then taken up in pH 5.0 buffer (300 mM acetic acid) andincubated at 37° C. overnight for 12–18 hours. The acidic pH of thebuffer cleaves the acetal linkage in the crosslinker moiety, producinglinear polymer chains and free DNA. The plasmid DNA was then quantifiedby fluorescence using PICOGREENTM intercalation (Molecular Probes,Eugene, Oreg.), a fluorescent dye that binds only double stranded DNA.

About 50% of the DNA that was originally loaded was encapsulated by themicrogels. Table 4 shows the estimated loading efficiency of themicrogels made with the bisacrylamide triglyme acetal crosslinker. Thehighest amount loaded was 4 μg DNA/mg of linear polymerizing group,however, the maximum amount of DNA that can be loaded into a sphere hasnot yet been reached.

TABLE 4 Supernatant Loading Estimated Concentration μg DNA/mg Efficiencyof Loading .μg/mL) microgel) encapsulation 2 μg DNA/mg bead 1.39 0.8844% 4 μg DNA/mg bead 1.31 2.14 53% 8 μg DNA/mg bead 1.87 4.29 54%

Analysis of Released DNA. There are three forms of plasmid DNA, which isnormally circular: 1) supercoiled, where the circular plasmid is furthercoiled; 2) open circular, where the plasmid has become untwisted but isstill circular; and 3) linear. Open circular and supercoiled plasmid DNAboth undergo transcription. When digested with HindIII and XmnIrestriction enzymes, the plasmid is cut twice into portions of 2263 bpand 4557 bp. The control was the plasmid DNA before transformation ofDH5αE. coli.

After hydrolytic release from the gel particles the DNA was subjected toa restriction digest with HindIII and XmnI and then analyzed by gelelectrophoresis (0.7% agarose, 50V for 150 minutes) and post stainedwith ethidium bromide (gel not shown). The lanes of control and releasedDNA subject to the double digest looked identical with some linearsingle cut plasmid still present. Prior to encapsulation, the DNA ismostly supercoiled (lower band at 4361 bp) with some open circular.After sonication, vacuum drying, and exposure to acidic solution for 18hours, the DNA is mostly open circular with some linear and supercoiledstructures.

Supercoiled and open circular plasmid DNA are still able to undergotranscription in cells but linear DNA cannot. When DNA is isolated frombacteria and loaded into the microgels, the DNA was mostly supercoiled.When isolated from the microgels, the DNA was mostly open circular, withsome linear and some supercoiled structure. The DNA at this point hadbeen through three major reaction conditions: sonication, radicalpolymerization, and acid exposure (pH 5, 37° C., 18 h). It is quiteremarkable that that the DNA remained intact because sonication of nakedDNA is known to shear and break it into linear strands or fragments. DNAis known to withstand acidic conditions, but it is rare to observe anykinetics after exposure to an acidic pH for such an extended period oftime. The restriction digest (single and double cut) serves to give afootprint, demonstrating that the DNA that was encapsulated and went into the polymerization had the same footprint as the DNA that wasrecovered.

Toxicity of DNA Encapsulated Microgel Particle. The microgels weretested for toxicity using the MTT assay of Example 14. RAW 309.CR1macrophage cells were split at 5×10⁴ cells per well in a 96 well plateand allowed to grow overnight. The cells were then incubated with themicrogel particles (1.6% crosslinked, sample A from Table 1 in Example11) with variable amounts of loaded DNA for 16 hours in DMEM media with10% F.B.S. The particles were aspirated from the cells, and they werethen washed several times with PBS and allowed to grow for another 48hours.

The cell viability was determined by measuring the absorbance of thereduced MTT reagent. The MTT assay was performed using 5 mg microgels/mLserum in each well with a particle loading of 0, 1, 2 and 4 μg DNA/mgmicrogels. After 48 hours, there were 82% viable cells remaining in theempty microgels, 70% viable cells remaining in the 1 μg DNA/mgmicrogels, 75% viable cells remaining in the 2 μg DNA/mg microgels, and65% viable cells remaining in the 4 μg DNA/mg microgels. Theconcentration tested (5 mg microgels/mL serum) is quite high for mostapplications, so a toxicity of 80% viability is permissible. It can beconcluded that neither the polyacrylamide microgels nor the DNA is toxicto the macrophage cells because at least 50% viable cells are left.

The microgel particles were also tested for DNA release. The microgelparticles were suspended in either pH 7.4 or pH 5.0 buffer. The amountof DNA released into the supernatant was quantified by fluorescenceusing PICOGREEN™ (Molecular Probes, Eugene, Oreg.). At pH 7.4, there isan initial burst, as is also seen with the protein loaded microgelparticles in Example 12. This is most likely due to DNA that is adsorbedonto the surface. At pH 5, all of the DNA is readily released within twohours. The microgels are visually degraded after 30 min and appear as agel in this assay after hydrolysis.

EXAMPLE 18 Evaluation of DNA Released from Microgels

Because the plasmid DNA is physically entrapped within the microgelparticles made with the bisacrylamide triglyme acetal crosslinker, theDNA is protected from otherwise being degraded in the serum. DNaseenzymes readily chew up naked DNA in serum, however the encapsulated DNAshowed good stability. The microgels were incubated in serum (90% DMEM,10% FBS) for a set period of time of 24 hours. The microgels were thencollected, and the serum supernatant removed. The plasmid DNA wasisolated from the microgel particles by placing in acetic acid for 6hours at, pH 5.0. Following hydrolysis of the microgels, the DNAreleased was quantified using PICOGREEN™. The DNA is fully protectedfrom enzymes in the serum as shown by the fact that transfection isstill possible with the recovered DNA.

The recovered DNA from the gel particles was tested for its viability bytransfecting cells. Kidney 293T cells (ATCC, Manassas, Va.) is a kidneycell line, relatively easy to transfect. The 293T cells are treated withDNA and Lipofectamine 2000 (Promega, Madison, Wis.), a cationic lipid, aknown transfection agent. If the DNA that is recovered is intact, thecells should produce β-galactosidase upon transfection. After 24 h, thecells were lysed and a galacto-ortho-nitrophenol substrate was added.β-galactosidase is present, the acetal bond in galacto-ortho-nitrophenolis cleaved and the released phenolate turns purple and absorbs at 570nm.

The addition of plasmid DNA alone to the cells causes no transfectionand no β-galactosidase activity is detected. Cells that were transfectedby the control plasmid DNA showed an absorbance of 0.55, 0.6 and 1.0 atDNA concentrations of 0.25 ng, 0.50 ng and 1.0 ng. Cells transfectedwith open circular DNA isolated from the microgel particles also showsβ-galactosidase activity and had absorbances of 0.4 and 0.55 at 0.25 ngand 0.50 ng respectively.

However, no transfection is observed with RAW cells, macrophages, eitherwith the known control plasmid DNA or with the DNA isolated from themicrogel particles, likely because macrophages are very difficult totransfect.

EXAMPLE 20

Evaluation of Immune Response to DNA Released from MicrogelParticlesPlasmid encapsulated microgels were fed to macrophages, and twoindicators of immune activity were analyzed by evaluating IL-6 and NO₂levels which are both indicative of an immunistimulatory response.

Interleukin-6 was detected by an ELISA assay. RAW 264.7 macrophages wereincubated with either microgels encapsulating plasmid DNA or nakedplasmid overnight. The supernatant was analyzed for IL-6 productionusing an ELISA kit (Pierce Biotechnology, Rockford, Ill.). The IL-6enzyme levels were detected for the following amounts of DNA added toeach well: untreated (control), 1 μg of DNA encoding β-galactosidase, 1μg plasmid DNA+lipofectamine, unloaded microgels, 0.1 μg plasmid DNA inmicrogels, 0.2 μg plasmid DNA in microgels, and 0.4 μg plasmid DNA inmicrogels.

Untreated cells as well as those incubated with plasmid DNA show a lowlevel of about 300–400 μg/mL of secreted IL-6. When DNA was mixed withLipofectamine 2000, the IL-6 level is increased to about 2000 μg/mL ofIL-6. Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, Calif.) is acommercially available transfection agent that forms micelles around theDNA, protecting it from nucleases in the serum and facilitating cellulardelivery. Therefore, this shows that naked DNA requires a transfectionagent, such as Lipofectamine, in order to produce an immune response.

Unloaded microgels had no IL-6 response, meaning that microgels alone donot induce an immune response. Microgels with DNA did induce IL-6secretion. When as little as 0.1 μg of DNA is delivered per well, animmune response of about 350 pg/ml of IL-6 is observed. When 0.2 μg ofplasmid DNA is delivered in microgels, there is about 1700 pg/mL of IL-6detected. This is equivalent to a 30-fold increase in IL-6 productionwhen compared to naked DNA alone, for which 1 μg is delivered per well.For a loading in which 0.2 μg of plasmid DNA is delivered per well usingthe microgels, an IL-6 response of 7 times that of DNA alone wasobserved where 5 times the amount of DNA is delivered. A higher loadingof DNA, 0.4 μg, does not produce a higher response, but a production of1500 pg/mL IL-6 is observed, indicating that a maximum response wasreached for DNA loading.

Macrophages also release other mediators including prostraglandins,oxygen radicals, peroxides, NO, etc. Comparing these same samples again,there was a 70-fold enhancement in NO activity when the DNA isencapsulated in the microgels. Nitric oxide is detected by the Griessassay by measuring NO₂ ⁻. RAW 309.1 cells were split onto a 96 wellplate at 4×10⁵ cells per well the night before the experiment and grownin 10% serum containing DMEM medium. The medium was then removed and theappropriate microparticle sample was added, and the cells were grown inserum containing media for 16 hours. The media was then aspirated offand the cells were stimulated with 10 units/ml of gamma interferon and10 μg/ml of LPS for 8 hours in serum containing media (to stimulate NO₂production). The medium was then isolated and the concentration of NOwas measured by mixing the supernatant with an equal volume of Griessreagent (1% sulfanilamide, 0.1% naphthylethylene-diamine hydrochloride,and 5% phosphoric acid). The absorbance at 540 nm was measured after 10minutes at room temperature.

There was no detected NO production for the untreated cells, or thecells incubated with 1 μg of DNA encoding β-galactosidase, unloadedmicrogels, or 0.1 μg plasmid DNA in microgels. About 14 μM of NaNO₂ wasdetected for cells incubated with 1 μg plasmid DNA+lipofectamine, 15.5μM of NaNO₂ was detected for cells incubated with 0.2 μg plasmid DNA inmicrogels, and 11 μM of NaNO₂ was detected for cells incubated with 0.4μg plasmid DNA in microgels.

EXAMPLE 21 Test particles with Dendritic Cells in In Vitro and In VivoStudies

In the in vitro experiment, bone marrow dendritic cells are grown thenpulsed with the microgel particles made according to Example 7.Dendritic cells phagocytose antigens and display antigens uponmaturation. Therefore these in vitro studies can show that the microgelsof the present inventions are effective in delivering bioactive materialto dendritic cells which then display them and initiate the innateimmune response.

These microgel particles are made with the crosslinkers of Example 4 or5. The dendritic cells are pulsed at an immature stage and then culturedwith ovalbumin (OVA) transgenic CD4 and CD8 T-cells for several days.The following groups can be tested: microgels entrapping OVA, microgelsentrapping OVA+TNF, microgels entrapping protein control, microparticlesentrapping protein control+TNF, OVA alone, OVA+TNF, and peptide+TNF. Theamount of OVA used should be about 50–100 μg/ml, which means thatapproximately 250–500 μg of protein total or less should be entrappedwithin the microgels. A similar amount of microgels that do not containOVA or that contains a different protein is required for a control.

In the in vivo experiment the microparticles are injected into the foodpad of CD4 or CD8 transgenic mice to show that microgels can activatecytotoxic T lymphocytes in vivo. More preferably, delivery is byinjection of 50 μl of resuspended particle using a 25 gauge syringe inthe flanks of these transgenic mice. At least 50 μg of OVA/mouse shouldsuffice per injection with at least 3 mice per group injected. Also 150μg of microgels with OVA and a similar amount of mirogels used forcontrol are injected. The lymph nodes are isolated 7 days after theinjection and analyzed for antigen specific T cell priming.

While the present compositions and processes have been described withreference to specific details of certain exemplary embodiments thereof,it is not intended that such details be regarded as limitations upon thescope of the invention, which should be regarded as defined by thefollowing claims.

1. An acid hydrolyzable microgel composition, comprising: (a) a polymerbackbone crosslinked by an acid hydrolyzable crosslinker, wherein saidcrosslinker hydrolyzes at pH 4.5 to pH 7.4; (b) the crosslinker havingthe formula R²CH(OR¹)₂, wherein R¹ is an acryloyl group; and R² is Ar—Xwhere X is a water solubilizing group selected from hydrogen, methoxy,—O—(CH₂—CH₂—O)_(n)—CH₃ wherein n is from 1 to 10,—O—CH₂—CH₂—O—C(O)—O—Ph—NO₂ and —O—CH₂—CH₂—CH₂ —NH—CO-(dextranpolysaccharide), said dextran polysaccharide having a molecular weightfrom 300 to 100,000 daltons; and Ar is an aryl group; (c) a particlesize of the microgel composition between 0.1–10 microns; and (d) crosslinkages between 1 and 20 mole percent.
 2. The composition of claim 1wherein R¹ is ethylacrylamide and R² is such that Ar is phenyl and X ismethoxy.
 3. The composition of claim 1 wherein R¹ is an acrylate,whereby crosslinker hydrolysis causes generation of further acidicspecies in an autocatalytic manner.
 4. The composition of claim 1wherein the particle size is between 200 nm and 500 nm.
 5. Thecomposition of claim 1 wherein said polymer backbone is comprised of adextran polysaccharide, said dextran polysaccharide having a molecularweight from 300 to 100,000 daltons.
 6. The composition of claim 1further comprising a bioactive material, wherein the bioactive materialis selected from the group consisting of palysaccharides, DNA, RNA,amino acids, and proteins.
 7. The composition of claim 6 whereby thebioactive material is physically entrapped within the microgelcomposition.
 8. The composition of claim 6 whereby the bioacdve materialis adsorbed onto the microgel composition.
 9. The composition of claim 6wherein said bioactive material is an antigen.
 10. The composition ofclaim 6 wherein the bioactive material is unmethylated DNA.
 11. An acidhydrolyzable polydextran microgel composition for delivering a bioactivematerial, comprising: (a) a polymerized and crosslinked acidhydrolyzable crosslinker of the formula R²CH(OR¹)₂, wherein saidcrosslinker hydrolyzes pH 5.0 to pH 7.4, wherein R¹ is an acryloylgroup, and R² is Ar—X where X is an alkyl dextran, wherein said dextranhas a molecular weight from 300 to 100,000 daltons; and Ar is an arylgroup; (b) a particle size of the polydextran microgel compositionbetween 0.1–10 microns; and (c) cross linkages between 1 and 20 molepercent.
 12. The composition of claim 11 wherein said alkyl-dextranlinker has the formula —O—CH₂—CH₂—O—C(O)—NH—[CH₂—CH₂—O]₂—CH₂—CH₂—NH—C(O)-dextran, wherein said dextran has a molecular weight from 300 to100,000 daltons.
 13. The composition of claim 11 further comprising abioactive material, wherein the bioactive material is selected from thegroup consisting of polysaccharides, DNA, RNA, amino acids, andproteins.
 14. The composition of claim 13 whereby the bioactive materialis physically entrapped within the microgel composition.
 15. Thecomposition of claim 13 whereby the bioactive material is adsorbed ontothe microgel composition.
 16. The composition of claim 13 wherein saidbioactive material is an antigen.
 17. The composition of claim 13wherein the bioactive material is unmethylated DNA.